Method for freeze drying a bacteria-containing concentrate

ABSTRACT

The present invention relates to a process for freeze drying a bacteria-containing concentrate. Further, the present invention relates to the freeze dried concentrates per se.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. National Stage of InternationalApplication No. PCT/EP2013/067338, filed on Aug. 20, 2013, and claimspriority to Danish Patent Application No. 201200513, filed on Aug. 20,2012, International Application No. PCT/EP2012/066208, filed on Aug. 20,2012, Danish Patent Application No. 201200821, filed on Dec. 21, 2012,and Danish Patent Application No. 201300101, filed on Feb. 20, 2013.

FIELD OF THE INVENTION

The present invention relates to an improved process for freeze drying abacteria-containing concentrate. Further, the present invention relatesto the novel freeze dried concentrates obtained by the process.

BACKGROUND OF THE INVENTION

Before inoculation into products, such as food products, bacteria arecultured in order to provide a suspension containing large amounts ofbacteria. The suspension is usually concentrated using centrifugation,filtration, distillation, sedimentation or flocculation. Thisconcentration step is often followed by freezing or freeze-drying ordrying or storage of the microbial concentrate as a frozen product inliquid nitrogen to preserve and/or store the bacteria.

However, freeze-drying of the bacteria is a bottleneck in the industrialproduction of storable viable bacteria due to the cell damage and lossof viable cells during the freeze-drying but also due to the longprocess time, usually days, thus resulting in high cost industrialfreeze drying processes. This is primary due to the primary drying, orice sublimation, stage of freeze drying process which is frequently themost time consuming portion of the process (PIKAL M. J.; SHAH S.). Thisis due to the practice to keep the products at a low product temperatureduring primary freeze-drying in order to avoid product collapse i.e.loss in particle microstructure which is regarded as determinable forproducts quality and stability (Schersch et al. 2256-78). Productcollapse is usually avoided commercially by applying mild processconditions i.e. low pressure and low shelf/heating plate temperatureduring freeze-drying, which results in prolonged drying cycles. Primarydrying starts when pressure in the freeze-drying cabinet is decreasedand shelf temperature is increased (Georges CORRIEU; Frangois-MarieLUQUET, 2008).

Therefore, there is still a need to improve the efficiency of freezedrying methods suitable for bacteria-containing suspensions, to obtain ahighly concentrated bacteria suspension with a limited loss ofbiological activity as well as a limited loss of viable bacteria. Thesemethods need to be feasible at any scale, but especially on theindustrial scale, where large volumes of suspension are concentrated.

SUMMARY OF THE INVENTION

The present inventors have researched intensely in ways to improve thefreeze-drying processes, and have now provided a novel process forfreeze drying of bacteria, said process involve use of a freeze dryercontaining means for heating the frozen bacteria containing concentrateto be dried, such as heating plates.

It has surprisingly turned out that it is possible to perform the freezedrying at higher temperature and/or higher pressure than conventionallyused, without negative impact on the cell viability, biological activity(acidification or other activity) and water activity of the resultingproduct, compared to a product produced under conventional conditions.Moreover, the tested more aggressive freeze drying conditions (highertemperature and/or higher pressure) also result in a faster and moreefficient drying, and thus in less costly manufacturing process.

Thus, in a first aspect, the invention relates to process for drying abacteria-containing concentrate (which optionally contains one or moreadditives, such as a cryo protectant and/or a stabilizer), comprising:

-   -   i) freezing said bacteria-containing concentrate; and    -   ii) freeze-drying said frozen bacteria-containing concentrate        obtained in step i); wherein step ii) is performed under the        following conditions:    -   a) a pressure in the range of 0.2 to 2.0 mBar, such as in the        range of 0.5 to 2.0 mBar; and    -   b) a heating plate temperature in the range of 30 to 100 degrees        C.

Further, it has surprisingly turned out that—besides addition of acryoprotectant—the purification of the bacteria cells has a huge impacton the viability of the bacteria after the freeze-drying step.Especially, it has surprisingly been discovered that the water activity(aw) of the bacteria obtained by the process is significantly lower thanexpected.

Thus, in a preferred embodiment of the first aspect thebacteria-containing concentrate has been washed before freezing.

The process can be used for improving the storage stability of abacteria-containing concentrate, more preferably a freeze-driedbacteria-containing concentrate. The process can also be used forimproving the viability of bacteria upon preservation by freezing orfreeze-drying.

In another aspect, the present invention relates to abacteria-containing concentrate obtainable by a process of the presentinvention. Preferred concentrates according to the invention containabout 10E9 to about 10E12 cfu/mL and/or are frozen or freeze-dried.Interestingly, such a freeze-dried bacteria-containing concentrate has alower water activity (aw) than a freeze-dried concentrate produced underidentical conditions, but with an unwashed concentrate as a startingmaterial.

DETAILED DISCLOSURE

Thus, in a first aspect, the present invention relates to a process fordrying a bacteria-containing concentrate which optionally contains oneor more additives, such as a cryo protectant and/or a stabilizer, saidprocess comprising:

i) freezing said bacteria-containing concentrate; and

ii) freeze-drying said frozen bacteria-containing concentrate obtainedin step i);

wherein step ii) is performed under the following conditions:

a) a pressure in the range of 0.2 to 2.0 mBar; and

b) a heating plate temperature in the range of 30 to 100 degrees C.

In a preferred embodiment, the optional additive is selected from thegroup consisting of inosine, inositol, IMP, trehalose, sucrose,maltodextrin and protein hydrolyzate.

Preferably, the pressure that is applied during the freeze-drying stepis in the range of 0.5 to 2.0 mBar, more preferably in the range 0.5 to1.0 mBar, 0.6 to 0.8 mBar, and even more preferably 0.5 to 0.6 mBar. Thepressure applied during the freeze-drying step may also be in the rangeof 0.8 to 1.5 mBar, 0.8 to 1.1 mBar, 0.7 to 1.1 mBar, 0.4 to 0.6 mBar,0.9 to 1.3 mBar, or 1.0 to 1.9 mBar. Further ranges for pressure (mBar)are: 0.1 to 2.0, 0.2 to 1.3, 0.2 to 1.2, 0.3 to 1.1, 0.3 to 1.1, 0.4 to1.0, 0.4 to 0.9, 0.5 to 0.8, 0.5 to 0.7, 0.9 to 2.0, 0.9 to 1.8, and 0.3to 0.7. The end-points of all the above ranges can be freely combined.

The pressure applied during the freeze-drying step is preferablymaintained for a certain time period, e.g. for more than 1 hour, morepreferably for more than 2, 3, 4, 5, or even more than 7 hours.

During the freeze-drying, it is particularly preferred to adjust thetemperature of the heating unit of the freeze-drying device, e.g. theheating plate, to a temperature in the range of 40 to 90 degrees C.,preferably 50 to 85 degrees C., more preferably 55 to 80 degrees C., andeven more preferably in the range 60 to 75 degrees C. Further suitableranges for temperature (degrees C.) are: 41 to 85, 41 to 59, 50 to 75,and 41 to 75. Also, the range 50 to 85 degrees C., (optionally excludingthe temperatures of 60, and 70 and 80 degrees C.) is a part of theinvention. The end-points of all the above ranges can be freelycombined. It has been proven particularly useful to maintain thistemperature of the heating unit, e.g. the heating plate, for more than 1hour, more preferably for more than 2, 3, 4, 5, or more than 7 hours.

It should be understood that the drying conditions as defined in thepresent invention may be applied during the entire drying process. Byway of example only, the entire drying process could be from 5 hours upto several days, e.g. three days. In a particular embodiment the dryingpressure as defined in the invention is applied until the pellets had anAw (water activity) below 0.10.

However in a particular embodiment the drying conditions as defined inthe invention is applied during only a part of the drying process, suchas for example during the first part, in the middle and/or at the end ofthe drying process. In a particular embodiment the drying pressure asdefined in the invention is applied for example during the first part,in the middle and/or at the end of the primary drying phase. The primarydrying phase can be defined as that part of the freeze drying processthat involves the sublimation of ice. In contrast, secondary dryinginvolves the desorption of bound water. In another embodiment, thedrying can start using a low standard pressure (e.g. 0.2 mBar) then thedrying pressure can be increased to a value as defined in the presentinvention. The drying pressure as defined in the invention can thereforebe applied during a period of from 5 to 100 percent of the drying time,preferably from 10 to 100 percent, preferably from 20 to 100 percent,preferably from 30 to 100 percent, preferably from 40 to 100 percent,preferably from 50 to 100 percent, preferably from 60 to 100 percent,preferably from 70 to 100 percent, preferably from 80 to 100 percent,preferably from 90 to 100 percent of the drying time. In a particularexample, the drying pressure as defined in the invention is appliedduring the entire drying process (100 percent of the drying time). Thedrying pressure as defined in the invention can therefore be appliedduring a period of from 5 to 100 percent of the primary drying phasetime, preferably from 10 to 100 percent, preferably from 20 to 100percent, preferably from 30 to 100 percent, preferably from 40 to 100percent, preferably from 50 to 100 percent, preferably from 60 to 100percent, preferably from 70 to 100 percent, preferably from 80 to 100percent, preferably from 90 to 100 percent of the primary drying phasetime. In a particular example, the drying pressure as defined in theinvention is applied during the entire primary drying phase (100 percentof the primary drying time).

In yet another embodiment, the drying can start using a relatively lowtemperature (e.g. −40 degrees C.) then the temperature can be increasedto a value as defined in the present invention. The drying temperatureas defined in the invention can therefore be applied during a period offrom 5 to 100 percent of the drying time, preferably from 10 to 100percent, preferably from 20 to 100 percent, preferably from 30 to 100percent, preferably from 40 to 100 percent, preferably from 50 to 100percent, preferably from 60 to 100 percent, preferably from 70 to 100percent, preferably from 80 to 100 percent, preferably from 90 to 100percent of the drying time. In a particular example, the dryingtemperature as defined in the invention is applied during the entiredrying process (100 percent of the drying time). The drying temperatureas defined in the invention can therefore be applied during a period offrom 5 to 100 percent of the primary drying phase time, preferably from10 to 100 percent, preferably from 20 to 100 percent, preferably from 30to 100 percent, preferably from 40 to 100 percent, preferably from 50 to100 percent, preferably from 60 to 100 percent, preferably from 70 to100 percent, preferably from 80 to 100 percent, preferably from 90 to100 percent of the primary drying phase time. In a particular example,the drying temperature as defined in the invention is applied during theentire primary drying phase (100 percent of the primary drying time).

The freeze dried concentrate may be further treated after freeze drying.In one aspect the dried concentrate is milled, but presently driedpellets are preferred.

It should be understood that in some freeze dryers the shelves of thefreeze dryer may constitute a heating unit, and in such a case the heatmight be applied directly to the shelf and the term heating platetemperature should be interchangeable with shelf temperature. In aspecific embodiment of the process of the present invention, the heatingplate temperature is equal to the temperature applied to theconcentrate.

In a specific embodiment, the drying process is performed until at least80%, preferably at least 90%, at least 95%, or at least 99%, of thewater is removed.

The drying process is preferably performed in a freeze-drying devicewhich is characterized by one or more of the following features:

-   a) the size of the freeze-dryer is such that it allows to process a    batch size of more than 100 kg; and/or-   b) the number of shelves and/or heating units, e.g. the heating    plates, is more than 5 and/or-   c) the thickness of the frozen bacteria-concentrate layer at the    time of initiation of the freeze drying process is in the range of 8    to 60 mm

A specific embodiment of the process of the invention is a process,wherein

-   -   a) the size of the freeze-dryer is such that it allows to        process a batch size of more than 100 kg; and/or    -   b) the number of shelves and/or heating plates is more than 10;        and/or    -   c) the thickness of the frozen bacteria-containing material on        the product trays from initiation of the freeze drying process        is 1 to 50 mm; and/or    -   d) the optional additive is selected from the group consisting        of: inosine, inositol, IMP, trehalose, sucrose, maltodextrin and        protein hydrolyzate.

Normally the concentrate is not placed directly on the shelves of thefreezedryer, but placed in a product tray. The concentrate can be placedin the freeze dryer in frozen form, such as pellets or a block, or theconcentrate can be placed in the freezedryer in form of a liquid orpaste, and subsequently frozen in situ.

In another preferred embodiment, the invention relates to an improvedfreeze-drying process for drying a bacteria-containing concentrate whichcontains inosine, inositol, trehalose and/or sucrose, skim milk, saidprocess comprising:

i) freezing said bacteria-containing concentrate; and

ii) freeze-drying said frozen bacteria-containing concentrate obtainedin step i);

wherein step ii) is performed under the following conditions:

a) a pressure in the range of 0.3 to 0.6 mBar; and

b) a heating plate temperature in the range of 40 to 80 degrees C.;

and wherein the conditions are kept for more than 1 hour, and/or untilat least 80% of the water is removed.

In yet another preferred embodiment, the invention relates to animproved freeze-drying process for drying a bacteria-containingconcentrate (e.g. containing one of more LAB strains) which containse.g. inosine, inositol, trehalose and/or sucrose, said processcomprising:

-   i) freezing said bacteria-containing concentrate; and-   ii) freeze-drying said frozen bacteria-containing concentrate    obtained in step i);-   wherein step ii) is performed under the following conditions:-   a) a pressure in the range of 0.5 to 2 mBar, such as in the range    0.6 to 1.8 mBar; and-   b) a heating plate temperature in the range of 30 to 100, such as in    the range 50 to 90 degrees C.;    and wherein the conditions are kept for more than 1 hour, and/or    until at least 75% (such as 80%) of the water is removed.

The bacteria-containing concentrate used in the improved freeze-dryingprocess comprises at least one LAB genus, preferably selected from thegroup consisting of Lactococcus, Lactobacillus, Leuconostoc,Carnobacterium, Pediococcus, and Streptococcus and more preferably atleast one species selected from the group consisting of Leuconostocspp., Bifidobacterium ssp, Lactococcus lactis, Lactococcus cremoris,Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefir,Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus,Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillussalivarius, Lactobacillus curvatus, Lactobacillus bulgaricus,Lactobacillus sake, Lactobacillus reuteri, Lactobacillus lactis,Lactobacillus delbreuckii, Lactobacillus plantarum, and Streptococcusthermophilus. It is particularly preferred that bacteria in thebacteria-containing concentrate belong to one of the above species.

Preferably, the bacteria-containing concentrate to by dried by use ofthe improved freeze-drying process is a “washed bacteria-containingconcentrate”. This “washed bacteria-containing concentrate” optionallycontains one or more additives, such as a cryo protectant and/or astabilizer as defined herein, which preferably has been added after thewashing process.

Finally, the invention also provides dry bacteria-containing concentratethat is obtainable by the improved freeze-drying process disclosedherein. Preferably, this bacterial concentrate comprises from 10⁹ to10¹³ cfu/g LAB cells.

In an interesting embodiment of the first aspect of the invention, thebacteria-containing concentrate has been prepared by a processcomprising the following steps:

-   B) adding an aqueous solution to a first bacteria-containing    concentrate to provide a bacteria-containing suspension, wherein the    volume of the added aqueous solution is in the range of 0.3 liters    to 10 liters per liter of said first bacteria-containing    concentrate;-   B1) optionally mixing;-   C) concentrating said bacteria-containing suspension to provide a    washed bacteria-containing concentrate; and-   C1) optionally repeating steps B), B1) and C);-   C2) optionally adding a cryoprotectant and/or a stabilizer;    or by a process comprising the following steps:-   A) providing a first bacteria-containing concentrate;-   B) adding an aqueous solution to said first bacteria-containing    concentrate to provide a bacteria-containing suspension, wherein the    volume of the added aqueous solution is in the range of 0.3 liters    to 10 liters per liter of said first bacteria-containing    concentrate;-   C) concentrating said bacteria-containing suspension to provide a    washed bacteria-containing concentrate.

In a further embodiment, the processes may further comprise a step (D),which is selected from one or more of the following:

i) recovering said second bacteria-containing concentrate; and/or

ii) freezing said second bacteria-containing concentrate.

In a preferred embodiment the invention thus relates to a process forimproving the storage stability of a bacterial concentrate, comprisingthe following steps:

-   -   A) providing a first LAB-containing concentrate;    -   B) adding an aqueous solution to said first LAB-containing        concentrate to provide a LAB-containing suspension, wherein the        volume of the added aqueous solution is in the range of 0.3        liters to 10 liters per liter of said first LAB-containing        concentrate;    -   B1) mixing;    -   C) concentrating said LAB-containing suspension to provide a        washed LAB-containing concentrate;    -   C1) optionally repeating steps B), B1) and C) at least one time;    -   C2) adding at least one cryo protectant and/or a stabilizer; and    -   D) freezing and/or freeze-drying said washed LAB-containing        concentrate.

The processes according to the present invention provide preferablybacteria-containing concentrates wherein the cell survival, i.e. thepercentage of active, i.e. viable, cells relative to the total cellnumber, is particularly high. It is e.g. preferred that at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, orabout 100% of the bacterial cells in the second bacterial concentrateare viable. The skilled person is aware of suitable methods to determinethe amount of cells in a concentrate that are viable. For example, flowcytometric methods may be used to quantify active and inactive, i.e.viable and non-viable, cells on the level of energy metabolism. Activecells are detected by a cellular fluorescence staining, whichdifferentiates cells generating a membrane potential from cells withoutsuch functional energy metabolism.

In an embodiment of the processes of the invention, the volume of theaqueous solution in step B) is in the range of 0.5 liter to 6 liters perliter of said first bacteria-containing concentrate, such as in therange of 1 liter to 4 liters, in the range of 1.5 liters to 3.5 liters,in the range of 1.0 liters to 3.0 liters, in the range of 1.0 liters to2.0 liters, in the range of 1.0 liters to 1.5 liters, in the range of1.5 liters to 2.0 liters, or in the range of 2 liters to 3 liters. It isparticularly preferred that the volume of the aqueous solution in stepb) is in the range of 0.5 liters to 2.0 liters per liter of said firstbacteria-containing concentrate. It is further preferred that the volumeof the aqueous solution in step B) is in the range of 1.0 liters to 2.0liters per liter of said first bacteria-containing concentrate. Further,the volume of the aqueous solution in step B) is preferably more than0.1 and equal to or below 2 liters per liter of said firstbacteria-containing concentrate

It is presently preferred that the concentrating in step C) is carriedout as a centrifugation step, said centrifugation step is preferablycarried out at a centrifugation force from about 400 to about 65000×g,preferably from about 4000 to about 20000×g, preferably from about 8000to about 15000×g. The skilled person will be able to choose thecentrifugal force such that only a minor portion of the bacteria in thepellet or concentrate will be rendered non-viable by the centrifugationstep(s). For example, after a centrifuging step according to theinvention preferably more than 50%, more than 60%, more than 70%, morethan 80% or more than 90% of the bacteria that were viable before thecentrifugation step was carried out remain viable.

The concentrating in step C) may also be carried out as a filteringstep, said filtering step comprises microfiltration, preferably using afiltration membrane having a pore size of 0.1 to 10 micrometres.Alternatively, said filtering step comprises ultrafiltration, preferablyusing a filtration membrane having a molecular weight cut-off of about 5to about 200 kDa. The filtering step may comprise tangential filtration,i.e. filtration wherein the concentrate tangentially flows across thesurface of the filtration membrane while the permeate flows through thefiltration membrane.

The first bacteria-containing concentrate to be used in step A) and/orB) has preferably been obtained by concentration of a fermentation brothor a culture containing the bacteria. In one embodiment of the processesof the invention, the first bacteria-containing concentrate may beobtained by centrifuging a fermentation broth, e.g. using a continuouscentrifuge. The first bacteria-containing concentrate may be in form ofa frozen concentrate, a liquid (ambient) concentrate, a pressedconcentrate (such as a filter cake), a dried concentrate, a spray driedconcentrate, a vacuum dried concentrate or in form of a freeze-driedconcentrate.

The first bacteria-containing concentrate is concentrated at a ratio inthe range 5 to 25 times, such as 10-20 times, based on the volume of thefermentation broth. Thus, preferably, the first bacteria-containingconcentrate has been concentrated 5- to 25-fold, preferably 10- to20-fold, more preferably about 15-fold, based on the volume of thefermentation broth.

In an alternative embodiment, the first bacteria-containing concentratemay be obtained by filtration of a fermentation broth, e.g. usingtangential filtration and/or ultrafiltration.

In a preferred embodiment, the first bacteria-containing concentrate isobtained by centrifuging a fermentation broth, and the concentrating ofthe bacteria-containing suspension in step C) is performed bycentrifugation. At least one of said centrifuging steps may be carriedout at a centrifugation force from about 400 to about 65000×g,preferably from about 4000 to about 20000×g, preferably from about 8000to about 15000×g.

It should be understood that a convenient implementation of the processof the invention is a continuous flow process. Thus, in a preferredembodiment, the concentrate to be frozen is the obtained directly from acontinuous centrifuge or cross flow filtration unit.

In a further embodiment, the washing and concentration processes mayfurther comprise the step of recovering the supernatant obtained in thecentrifuging step and/or the permeate obtained in the filtering step.

The pH of the aqueous solutions of step B) may be in the range of 3 to8, such as in the range 4 to 7 or in the range 3 to 7.

The temperature of the aqueous solution of step B) may be in the rangeof 0 to 50 degrees C. (° C.), such as in the range 3 to 30 degrees, inthe range of 5 to 25 degrees, or in the range of 10 to 20 degrees C.

In a further embodiment, one or more, preferably all, of the steps ofthe washing and concentration processes are carried out at a temperaturein the range of 0 to 50 degrees C., such as in the range 3 to 30 degreesC., in the range of 5 to 25 degrees C., or in the range of 10 to 20degrees C.

In an preferred embodiment, the bacteria-containing concentrate in theprocesses according to the present invention is a LAB-containingconcentrate. Such a bacteria-containing concentrate may comprise abacteria selected from the group consisting of Acetobacter,Bifidobacterium, Carnobacterium, Enterococcus, Lactococcus,Lactobacillus, Leuconostoc, Pediococcus, Oenococcus, Propionibacterium,and Streptococcus.

More specifically, the bacteria-containing concentrate may comprise atleast one strain of a LAB genus, preferably selected from the groupconsisting of Lactococcus, Lactobacillus, Leuconostoc, Carnobacterium,Pediococcus, and Streptococcus and more preferably at least one strainof a species selected from the group consisting of Leuconostoc spp.,Bifidobacterium ssp, Lactococcus lactis, Lactococcus cremoris,Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefir,Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus,Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillussalivarius, Lactobacillus curvatus, Lactobacillus bulgaricus,Lactobacillus sake, Lactobacillus reuteri, Lactobacillus lactis,Lactobacillus delbreuckii, Lactobacillus plantarum, and Streptococcusthermophilus.

In a presently preferred embodiment of the first aspect, the bacteria isof a strain selected from the group consisting of BB-12® that wasdeposited with the German Collection of Microorganisms and Cell Cultures(DSMZ) under the accession no. DSM15954, BB-12® free that was depositedwith DSMZ under the accession no. DSM17281, LA-5° that was depositedwith DSMZ under the accession no. DSM13241, ST6008 that was depositedwith DSMZ under the accession no. DSM18111, LGG® that was deposited withthe American Tissue type Collection Center under the accession no.ATCC53103, ST-4895 that was deposited with DSMZ under the accession no.DSM19242 and R-607-1 that was deposited with DSMZ under the accessionno. DSM21404, and/or mutants or variants thereof.

Streptococcus thermophilus strain ST10255 is commercially available fromChr. Hansen A/S as F-DVS ST-BODY-2, Material No: 623155.)

In the present context, the term “mutant” should be understood as astrain derived from a mother strain by means of e.g. geneticengineering, radiation, UV light, and/or chemical treatment and/or othermethods that induce changes in the genome. It is preferred that themutant is a functionally equivalent mutant, e.g. a mutant that hassubstantially the same, or improved, properties as the mother strain.Such a mutant can be used in the processes according to the presentinvention. Especially, the term “mutant” refers to a strain obtained bysubjecting a strain to any conventionally used mutagenization treatmentincluding treatment with a chemical mutagen such as ethane methanesulphonate (EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UV lightor to a spontaneously occurring mutant. A mutant may have been subjectedto several mutagenization treatments (a single treatment should beunderstood as containing one mutagenization step followed by ascreening/selection step), but it is presently preferred that no morethan 20, or no more than 10, or no more than 5 treatments (orscreening/selection steps) are carried out. In a presently preferredmutant, less that 5%, or less than 1% or even less than 0.1% of thenucleotides in the bacterial genome have been shifted with anothernucleotide, or deleted, compared to the mother strain. In the presentcontext, the term “variant” should be understood as a strain which isfunctionally equivalent to a strain of the invention, e.g. havingsubstantially the same, or improved, properties e.g. regardingviscosity, gel stiffness, mouth coating, flavor, post acidification,acidification speed, etc, when used for producing a dairy product. Suchvariants, which may be identified using appropriate screeningtechniques, are a part of the present invention.

The number of bacteria in the first bacteria-containing concentrate ofstep B) may be from about 10⁸ to about 10¹³ cfu/mL, such as from 10⁸ to10¹³ cfu/mL, from 10⁹ to 10¹³ cfu/mL, from 10⁹ to about 10¹² cfu/mL, orfrom 10¹⁰ to 10¹² cfu/mL.

The number of bacteria in the second bacteria-containing concentrate ofstep C) may be from about 10⁸ to about 10¹³ cfu/mL, such as from 10⁸ to10¹³ cfu/mL, from 10⁹ to 10¹³ cfu/mL, from 10⁹ to about 10¹² cfu/mL, orfrom 10¹⁰ to 10¹² cfu/mL.

The aqueous solution may be selected from the group consisting of:

-   -   Water, such as demineralized or destilled water;    -   An aqueous buffer;    -   A solution or suspension comprising 0 to 30% carbohydrate(s), 0        to 30% polysaccharide(s), 0 to 10% inorganic salt(s), 0 to 10%        organic salt(s), 0 to 10% protein(s) and/or peptide(s) and/or        amino acid(s), 0 to 10% cryoprotectant(s), 0 to 10%, water up to        100%;    -   Water containing from 1 to 300 g/l of a carbohydrate, such as        sucrose and/or trehalose;    -   water containing 1 to 100 g/l of an inorganic salt, such as        chloride or phosphate salts of sodium, potassium, calcium,        magnesium;    -   Water containing amino acids, carboxylic acids, inorganic acids        or inorganic bases;    -   Water containing carbohydrates wherein suitable carbohydrates        include saccharides such as one or more monosaccharides such as        dextrose; one or more disaccharides such as sucrose, trehalose        or lactose, one or more oligosaccharides, and/or one or more        polysaccharides such as dextran, mannan);    -   Water containing a buffer selected from the group consisting of        a carboxylic acid such as citric acid, an inorganic acid such as        phosphate buffer, an inorganic base, and a conjugate base/acid        thereto;    -   Water containing an alcohol wherein suitable alcohols include a        polyhydric alcohol such as a diol and a derivative thereof such        as e.g. polyethylene glycol, a triol such as e.g. glycerol, a        polyalcohol, and a sugar alcohol such as e.g. sorbitol;    -   Water containing a protein, a phosphoprotein such as e.g.        casein, a peptide, a peptone, yeast extract, malt extract,        tryptone, casein peptone, milk, skim milk, a milk buffer, and/or        milk powder;    -   Water containing a surfactant, such as a non-ionic surfactant        such as e.g. Tween 80;    -   Water containing an antioxidant such as an ascorbic acid; and    -   Water containing a vitamin or a trace element; and any        combination of the above solutions.

In some embodiments, a process for providing the bacteria-containingconcentrate may be a continuous flow process comprising: continuouslywithdrawing the first bacteria-containing concentrate duringcentrifugation; and continuously withdrawing the secondbacteria-containing concentrate during centrifugation. Nevertheless, theprocess of the invention may also be carried out as a batch process. Insome yet additional embodiments, the centrifuging is carried out at acentrifugation force from about 400 to about 65000×g. In somealternative embodiments, the centrifuging is carried out at acentrifugation force from about 4000 to about 20000×g.

As described above, the concentrating in step C) may also be carried outas a filtering step, such as a microfiltration and/or an ultrafiltrationstep. In some embodiments, a filtering step comprises microfiltration.In some still further embodiments, a filtering step utilizes afiltration membrane having a pore size of about 0.1 to about 10micrometres, preferably 0.2 to 5 micrometres, more preferably 0.5 to 2micrometres. Such microfiltration membranes typically have a molecularweight cut-off of about 200 kDa to about 5 000 kDa. In furtherembodiments, a filtering step comprises ultrafiltration. In some stillfurther embodiments, an ultrafiltration step utilizes a filtrationmembrane having a molecular weight cut-off of about 5 to about 200 kDa,preferably 10 to 100 kDa, more preferably 40 to 80 kDa. In somealternative embodiments, filtering comprises tangential filtration.

In some processes for providing the bacteria-containing concentrate, thefiltration step is accomplished using microfiltration with amicrofiltration membrane having a pore size from about 0.1 to about 10micrometres, while in other embodiments the pore size is from about 0.1to about 5 micrometres, and in still other embodiments, the pore size isfrom about 0.1 to about 2 micrometres. In some preferred embodiments,the microfiltration membrane has a molecular weight cut-off of about 200kDa to about 5 000 kDa, while in other embodiments, the molecular weightcut-off is about 250 kDa to about 1 000 kDa, and in still otherembodiments, the molecular weight cut-off is about 500 kDa.

In some alternative processes for providing the bacteria-containingconcentrate, the filtration step is accomplished using ultrafiltrationwith an ultrafiltration membrane having a pore size from about 0.01micrometres to about 0.1 micrometres, while in other embodiments, thepore size is from about 0.02 to about 0.1 micrometres, and in stillother embodiments, the pore size is from about 0.05 to about 0.1micrometres. In some embodiments, the ultrafiltration membrane has amolecular weight cut-off of about 5 kDa to about 200 kDa, while in otherembodiments, the molecular weight cut-off of is from about 30 kDa toabout 200 kDa, and in still further embodiments, the molecular weightcut-off is about 150 kDa.

The process for providing the bacteria-containing concentrate maycomprise a step (D) (i) wherein said second bacteria-containingconcentrate is recovered. Thus, in some further embodiments, theprocesses further comprise the step of recovering the supernatant and/orpermeate obtained after the centrifuging step and/or the filtering step.

The processes provide means for the rapid and efficient concentration ofbacteria-containing suspensions. Importantly, the processes of thepresent invention find use on an industrial scale, as significantvolumes of bacteria-containing suspensions can be treated. In someembodiments, the processes of the present invention find use in treatingsuspensions of about 500 L to about 100,000 L. In some embodiments, thesuspensions range from about 10,000 L to about 50,000 L, while in otherembodiments, the range is from about 10,000 L to about 25,000 L.However, it is not intended that the present invention be limited tothese volumes, as it is contemplated that any suitable volume ofbacteria-containing suspension will find use in the processes of thepresent invention.

Furthermore, the processes of the present invention facilitateeconomical and efficient additional processing of the bacterialsuspensions. For example, because the water content of concentrate islower and the bacteria concentration is higher than suspensions producedusing other processes, freeze-drying and lyophilisation of thesesuspensions is accomplished faster and more efficiently than by usingstandard processes. Thus, by providing more rapid, efficient andeconomical means to preserve and store cultures, the present inventionprovides for reductions in energy, materials, and transportation costs.

In some embodiments, a process of the present invention involves atleast two washing steps. Thus, in these embodiments a step C1) iscarried out wherein the steps B), B1) and C) are repeated at least once.

In another embodiment, the process comprises a washing step followed bya centrifugation step.

In some further embodiments, the supernatant obtained after acentrifugation step(s) and/or the permeate obtained after a filtrationstep(s) are recovered. These embodiments find particular use whenmolecules of interest are present in the supernatant and/or in thepermeate. Some examples of molecules of interest include, but are notlimited to bacteriocins, enzymes and lactic acid. In some embodiments,the supernatant obtained after a centrifugation step(s) and/or thepermeate obtained after a filtration step(s) are subject to multiplecentrifugation and/or filtration step(s) before the recovery of themolecule(s) of interest.

As indicated above, in some embodiments, the processes of the presentinvention further comprise preservation and/or storage steps. In someembodiments, freeze-drying, drying, freezing and/or cooling find use. Itis not intended that the present invention be limited to any particularpreservation and/or storage steps. Also, a packaging step may beincluded in the process of the invention. In the present context, theterm “packaging” (a suitable amount of dried concentrate in a suitablepackage) relates to the final packaging of the concentrate to obtain aproduct that can be distributed to e.g. a person or a dairy. A suitablepackage may thus be a pouch of box or similar, and a suitable amount maybe e.g. 1 g to 10000 g, but it is presently preferred that the amount ina package is from 1 g to 1000 g.

The processes of the present invention find use in providing a highlyconcentrated concentrate of any suitable bacteria.

In some embodiments, the bacteria-containing suspension is afermentation broth containing bacteria. In some preferred embodiments,the bacteria comprise one or more of the genera Bifidobacterium,Lactobacillus, Streptococcus, and Lactococcus. However, it is notintended that the present invention be limited to any particular genusor species of bacteria.

As indicated herein, the process of the present invention areparticularly useful and cost effective for providing bacteria-containingconcentrates on an industrial scale, where large volumes are treated(e.g., about 500 L to about 100,000 L).

In some embodiments, the centrifugation step(s) considerably reduces thevolume of suspension, resulting in a concentrate. The centrifugationstep(s) may result in concentration rates of about 5 to about 25.

It is clear to the skilled person that a centrifugation step may bereplaced by another concentrating unit operation, e.g. a filtrationstep, convenient continuous filtration, such as tangential (cross-flow)ultra filtration. Also, the cell wash may be integrated in aconcentration step, e.g. if the second bacteria containing concentrateis obtained by means of tangential filtration, it is contemplated thatthe aqueous solution may be added during the filtration process. In sucha case, it is further contemplated that the aqueous solution may beadded in 50% of the amount needed when the second bacteria containingconcentrate is obtained by means of continuous centrifugation. Thus, thefirst aspect of the invention also relates to a process comprising thefollowing steps:

-   B) adding an aqueous solution to a first bacteria-containing    concentrate to provide a bacteria-containing suspension, wherein the    volume of the added aqueous solution is in the range of 0.15 liters    to 5 liters per liter of said first bacteria-containing concentrate;-   C) concentrating said bacteria-containing suspension to provide a    second bacteria-containing concentrate; and-   C1) optionally repeating steps B), and C);

C2) optionally adding a cryo protectant and/or a stabilizer;

wherein steps B) and C) are combined in a tangential flow filtrationprocess;

and to a process comprising the following steps:

-   B) adding an aqueous solution to said first bacteria-containing    concentrate to provide a bacteria-containing suspension, wherein the    volume of the added aqueous solution is in the range of 0.15 liters    to 5 liters per liter of said first bacteria-containing concentrate;-   C) concentrating said bacteria-containing suspension to provide a    second bacteria-containing concentrate;    wherein steps B) and B) are combined in a tangential flow filtration    process. Step B) may be preceded by a step A) which includes    providing a first bacteria-containing concentrate.

It should be understood that these steps are succeeded by the steps ofthe process claims.

In such a process, the volume of the aqueous solution may be in therange of 0.25 liter to 3 liters per liter of said firstbacteria-containing concentrate, such as in the range of 0.5 liter to 2liters, in the range 0.75 liters to 1.75 liters, in the range 0.5 litersto 1.5 liters, in the range 0.5 liters to 1.0 liter, in the range 0.5liters to 0.75 liters, in the range 0.75 liters to 1.0 liter, or in therange 1.0 liter to 1.5 liters.

An interesting embodiment is a process comprising the following steps:

-   B) washing said first bacteria-containing concentrate by adding an    aqueous solution to said concentrate to provide a    bacteria-containing suspension, wherein the volume of the added    aqueous solution is in the range of 1.0 liters to 3.0 liters,    preferably 0.5 liters to 2.0 liters, per liter of said first    bacteria-containing concentrate;-   C) concentrating said bacteria-containing suspension by    centrifugation to provide a second bacteria-containing concentrate;    and-   D) drying said second bacteria-containing concentrate by    freeze-drying.

Another interesting embodiment is a process comprising the followingsteps:

-   A) concentrating a fermentation broth by centrifugation, providing a    first bacteria-containing concentrate, preferably a LAB-containing    concentrate;-   B) washing and concentrating said first bacteria-containing    concentrate, to providing a second bacteria-containing concentrate,    using tangential ultrafiltration and before and/or during said    ultrafiltration adding an aqueous solution to said first    bacteria-containing concentrate, wherein the volume of the added    aqueous solution is in the range of 0.5 liters to 3.0 liters per    liter of said first bacteria-containing concentrate; and;-   D) drying said second bacteria-containing concentrate by    freeze-drying.

In the two preceding embodiments, it should of course be understood thatmore washing steps, more centrifugation steps, and/or more filtrationsteps may be added.

Other embodiments of the first aspect of the invention are:

-   -   The process of the invention, wherein the pressure is in the        range 0.2 to 2.0 mBar and the plate temperature is in the range        50 to 85 degrees C.    -   The process of the invention, wherein the pressure is in the        range 0.5 to 2.0 mBar and the plate temperature is in the range        50 to 85 degrees C.    -   The process of the invention, wherein the pressure is in the        range 0.2 to 1.3 mBar and the plate temperature is in the range        30 to 100 degrees C.    -   The process of the invention, wherein the pressure is in the        range 0.5 to 1.3 mBar and the plate temperature is in the range        30 to 100 degrees C.    -   The process of the invention, wherein the pressure is in the        range 0.4 to 0.8 mBar and the plate temperature is in the range        30 to 100 degrees C.    -   The process of the invention, wherein the pressure is in the        range 0.4 to 0.8 mBar and the plate temperature is in the range        40 to 90 degrees C.    -   The process of the invention, wherein the pressure is in the        range 0.4 to 2.0 mBar and the plate temperature is in the range        45 to 80 degrees C.    -   The process of the invention, wherein the bacteria-containing        concentrate contains at least two different LAB strains, such as        at least 3 or at least 4, or even at least 5 different LAB        strains.    -   The process of the invention, wherein the bacteria-containing        concentrate contains at least two different LAB strains, such as        at least 3 or at least 4, or even at least 5 different strains,        at least two of said strains belong to the same LAB species.    -   The process of the invention, wherein the bacteria-containing        concentrate contains at least two different LAB strains, such as        at least 3 or at least 4, or even at least 5 different strains,        at least two of said strains belonging to different LAB species.    -   The process of the invention, wherein the bacteria-containing        concentrate contains a strain of the species Streptococcus        thermophilus, and a strain of the species Lactobacillus        bulgaricus.    -   The process of the invention, wherein the bacteria-containing        concentrate contains a strain of a Leuconostoc species and a        strain of Lactococcus species.    -   The process of the invention, wherein the bacteria-containing        concentrate contains a strain of a Bifidobacterium species and a        strain of a Lactobacillus species.    -   The process of the invention, wherein the bacteria-containing        concentrate contains a strain of a Lactococcus species.    -   The process of the invention, wherein the bacteria-containing        concentrate contains a strain of a Lactobacillus helveticus        species.

In a second aspect, the present invention relates to a (dry or dried)bacteria-containing concentrate obtainable by the processes of theinvention. Such a concentrate may have a bacteria concentration fromabout 10⁹ to about 10¹² cfu/mL. Also, such a concentrate may have abacteria concentration from about 10⁹ to about 10¹³ cfu/g.

In an embodiment, the bacteria-containing concentrate according to theinvention may be in form of a freeze-dried concentrate that has lowerwater activity (a_(w)) than a freeze-dried concentrate produced underidentical conditions, but with an unwashed concentrate.

In another embodiment, the bacteria-containing concentrate of theinvention has been freeze-dried at a higher vacuum than possible for anunwashed concentrate, such as at a pressure in a range of 0.2-1.2 mBar,such as in the range of 0.3-0.9 mBar.

It yet another embodiment of the second aspect of the invention, thebacteria-containing concentrate may be in form of a freeze-driedconcentrate that has a higher purity and/or whiter colour than thefreeze-dried concentrate produced under identical conditions, but withan unwashed concentrate.

In a further embodiment, the bacteria-containing concentrate may be inthe form of a freeze-dried concentrate that is easier to grind than thefreeze-dried concentrate produced under identical conditions, but withan unwashed concentrate.

In some embodiments, the bacteria-containing concentrate contains a highnumber of bacteria per volume, at least about 10⁹ to about 10¹² cfu/mL,while in other embodiments, the final concentration is at least about5.10⁹ to about 9.10¹¹ cfu/mL.

The second aspect of the invention comprises a dry bacteria-containingconcentrate obtainable by a process of the invention. Interestingembodiments are:

-   -   A concentrate of the invention, which contains from 10⁹ to 10¹³        cfu/g of LAB cells.    -   A concentrate of the invention, which contains more than 1011        cfu/g.    -   A concentrate of the invention, which contains at least two        different LAB strains, such as at least 3 or at least 4, or even        at least 5 different LAB strains.    -   A concentrate of the invention, which contains at least two        different LAB strains, such as at least 3 or at least 4, or even        at least 5 different strains, at least two of said strains        belong to the same LAB species.    -   A concentrate of the invention, which contains at least two        different LAB strains, such as at least 3 or at least 4, or even        at least 5 different strains, at least two of said strains        belonging to different LAB species.    -   A concentrate of the invention, which contains a strain of the        species Streptococcus thermophilus, and a strain of the species        Lactobacillus bulgaricus.    -   A concentrate of the invention, which contains a strain of a        Leuconostoc species and a strain of Lactococcus species.    -   A concentrate of the invention, which contains a strain of a        Bifidobacterium species and a strain of a Lactobacillus species.    -   A concentrate of the invention, which contains a strain of a        Lactococcus species.    -   A concentrate of the invention, which contains a strain of a        Lactobacillus helveticus species.

In a third aspect, the present invention relates to the use of theprocess of the invention to prepare a freeze dried micro-organismcomposition having improved storage stability.

In a forth aspect, the present invention relates to the use of theprocess of the invention to prepare a freeze dried micro-organismcomposition having improved cell survival.

In a fifth aspect, the present invention relates to the use of theprocess of the invention to prepare a freeze dried micro-organismcomposition having lowered water activity (aw).

In a sixth aspect, the present invention relates to the use of theprocess of the invention to prepare a freeze dried micro-organismcomposition having a water activity (aw) below 0.3, below 0.15, below0.10 or below 0.08.

In a seventh aspect, the present invention relates to the use of theprocess of the invention to reduce freeze drying time.

In an eighth aspect, the present invention relates to the use of abacterial concentrate of the invention in the production of a dairyproduct, such as a cheese or a fermented milk product (e.g. yoghurt).

In a ninth aspect, the present invention relates to the use of abacterial concentrate of the invention in the production of a probioticor dietary supplement, e.g. to be administered to an animal, inclusive ahuman being.

The concentrates find use in various applications, including, but notlimited to food production, feed production, pharmaceutical production(e.g. as active ingredient in health beneficial probiotic products),etc. As indicated above, the present invention finds use in providingconcentrates of any suitable bacteria.

As indicated above, a process of the present invention provides means toobtain the desired final concentration of bacteria. The activity levelof the bacteria concentrates is directly linked to the number of viablebacteria. In some embodiments, the activity of the bacteria (i.e., themicrobial activity level) is determined by assessing the amount ofmetabolite(s) the culture produces over a given time period andutilizing a specific type of substrate. For example, for LAB, it ispossible to determine the activity level by continuously recording thepH for a given period of time, as the pH of a LAB culture is directlylinked to the concentration of viable bacteria. In some embodiments,comparing the recorded pH measurement to an expected theoretical pHvalue based on the assumption that all of the bacteria in the cultureare viable, provides the concentration and activity level of thesuspension. Thus, if the measured pH is close to the theoretical value,the bacterial population has undergone limited activity loss during theprocess.

The improved freeze-drying process of the present invention shallpreferably not include any one of the following embodiments:

-   A) LGG® (ATCC 53103) dried under a pressure of 0.2, 0.7, 1.3, 2.0,    or 2.5 mBar at a heating unit and/or heating plate and/or shelf    temperature of 50 degrees C.,-   B) LGG® (ATCC 53103) dried under a pressure of 0.2, or 1.3 mBar at a    heating unit and/or heating plate and/or shelf temperature of 60    degrees C., and/or-   C) LGG® (ATCC 53103) dried under a pressure of 0.2 or 1.3 mBar at a    heating unit and/or heating plate and/or shelf temperature of 70    degrees C.

In a preferred embodiment, these embodiments are disclaimed from thescope of the present invention.

Definitions

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although any processand materials similar or equivalent to those described herein find usein the practice of the present invention, the preferred process andmaterials are described herein. Accordingly, the terms definedimmediately below are more fully described by reference to thespecification as a whole. It is to be understood that the presentinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context inwhich they are used by those of skill in the art.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.Furthermore, the citation of any document is not to be construed as anadmission that it is prior art with respect to the present invention.

A “washing step” as used herein refers a process step in which thebacteria-containing concentrates are admixed with an aqueous solution.Thus, a washing step normally comprises the addition of an aqueoussolution to a bacteria-containing concentrate according to theinvention. A washing step may additionally comprise removal of a part ofthe previously added aqueous solution, such as e.g. by centrifugation orfiltration. Washing steps in the sense of the present invention may becarried out successively, e.g. by several successive addition andremoval steps in a continuous flow process (see below).

As used herein, the term “centrifugation” refers to a method ofseparating immiscible liquids or solids from liquids through applicationof centrifugal force. In some preferred embodiments, the separationmethods involve subjecting a fluid-containing mixture to a highgravitational force (g). Upon application of this centrifugal force(often many times the force of gravity [×g]), the different componentspresent in the mixture are separated. In some preferred embodiments ofthe present invention, centrifugation is applied to a liquid containingmicrobial cells. At the completion of the centrifugation process, themicrobial cells are located in the “concentrate” (i.e., the more “solid”portion of the product) and the liquid is the “supernatant” (or“eluate”). In some preferred embodiments, the liquid portion contains noor very few bacteria. In industrial scale volumes, the concentratetypically contains from between about 5 percent and about 20 percentsolids.

In some preferred embodiments, centrifugation is carried out in acentrifuge that provides a gravitational force from about 400 to about65000×g (i.e., times gravity), while in other embodiments, thegravitational force is from about 4000 to 20000×g, and in otherembodiments, the gravitational force is from about 6000 to about10000×g. However, it is not intended that the present invention belimited to any particular centrifuge or gravitational force. In somefurther embodiments of the present invention the centrifugation step isrepeated between about two and about four times. In some particularlypreferred embodiments, the centrifugation step is repeated twice.However, it is not intended that the present invention be limited to anyparticular number of repetitions of the centrifugation step, as anysuitable number of repetitions will find use.

As used herein, the term “filtration” refers to a separation processconsisting of passing a solution through a filtration membrane toseparate the components within the liquid, based on the size of thecomponents. The filtration membrane has pores of a given size designedto retain components that are larger than the pore size, but allowcomponents that are smaller than the pore size to pass through themembrane. Thus, in some preferred embodiments, the solution containssolid elements (e.g., bacteria) that are larger than the pores of thefiltration membrane. In these embodiments, the bacteria are present inthe “concentrate” (or “retentate”) and the liquid phase that passesthrough the membrane is referred to as “permeate” or “filtrate”. Inaddition to containing liquid, in some embodiments, the permeate alsocontains other components. In some embodiments of a process of thepresent invention, the filtration step results in the production of the“second bacteria-containing concentrate”. In “conventional filtration”the separation is carried out due to natural gravitational pressure,while in “pressure filtration,” additional pressure (e.g., greaterpressure on the concentrate side and/or a depression on the permeateside) helps to accelerate the filtration process. Any suitablefiltration methods find use in the present invention, including but notlimited to microfiltration and ultrafiltration. However, in someparticularly preferred embodiments, ultrafiltration is used.

As used herein, the term “micro filtration” refers to any filtrationmethod that involves use of microporous filtration membranes. The poresize of these microfiltration membranes is usually about 0.1 micrometresto about 10 micrometres, preferably 0.2 to 5 micrometres, morepreferably 0.5 to 2 micrometres. The microfiltration membranes used inthe methods of the present invention typically have a molecular weightcut-off of about 200 kDa to about 5 000 kDa.

As used herein, the term “ultrafiltration” refers to any filtrationprocess using filtration membranes having smaller pore sizes than thoseused for microfiltration, usually about 0.01 micrometres to about 0.1micrometres, preferably 0.04 to 0.08 micrometres. The ultrafiltrationmembranes used in the processes of the present invention typically havea molecular weight cut-off of about 5 kDa to about 200 kDa, preferably10 to 100 kDa, more preferably, 40 to 80 kDa.

It is intended that any suitable filtration method will find use in thepresent invention, including but not limited to conventional filtrationmethods (e.g., by use of gravitational force) and tangential orcross-flow filtration methods. The term “cross flow filtration” and“tangential filtration” are used interchangeably herein in reference toany filtration method wherein the concentrate continuously andtangentially flows across the surface of the filtration membrane whilethe permeate flows through the filtration membrane.

In continuous flow processing, the centrifuge is continuously fed withthe bacteria-containing suspension providing a continuous output flow offirst bacteria-containing concentrate. In the processes of the presentinvention, a concentration step may be combined with a washing step,e.g. by using diafiltration.

The “water activity” a_(w) as used herein is defined as the vaporpressure of water in the substance (e.g. the bacteria-containingconcentrate of the invention), divided by the vapor pressure of purewater at the same temperature. The skilled person is aware of numerousmethods to determine the water activity of a given substance. Forexample, a_(w) may be determined by measuring the vapor pressure in thesubstance and comparison of this vapor pressure with that of water atthe same temperature.

As used herein, the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid, acetic acid and propionic acid. Theindustrially most useful LAB are found within the order“Lactobacillales” which includes Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp.,Pediococcus spp., Brevibacterium spp., Enterococcus spp. andPropionibacterium spp. Additionally, lactic acid producing bacteriabelonging to the group of the strict anaerobic bacteria, bifidobacteria,i.e. Bifidobacterium spp., are generally included in the group of LAB.These are frequently used as food cultures alone or in combination withother LAB.

Preferably, the LAB are LAB selected from the group consisting of: LABbelonging to genus Lactobacillus, such as Lactobacillus helveticus,Lactobacillus delbruekii subsp. bulgaricus, Lactobacillus fermentum,Lactobacillus salivarius or Lactobacillus rhamnosus; LAB belonging togenus Lactococcus, such as Lactococcus lactis; LAB belonging to genusStreptococcus, such as Streptococcus thermophilus; LAB belonging togenus Leuconostoc, such as Leuconostoc lactis or L. mesenteroides; LABbelonging to genus Bifidobacterium, such as Bifidobacterium longum,Bifidobacterium animalis, or Bifidobacterium breve; LAB belonging togenus Propionibacterium; LAB belonging to genus Enterococcus, such asEnterococcus faecum; and LAB belonging to genus Pediococcus.

Even more preferably, the LAB are LAB selected from the group consistingof:

Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacteriumanimalis, Streptococcus thermophilus and Lactococcus lactis.

Most preferably, the LAB are LAB selected from the group consisting of:

Lactobacillus acidophilus, LA-5° (DSM13241),

Bifidobacterium animalis, BB-12® (DSM15954),

Lactobacillus rhamnosus, LGG® (ATCC53103),

Streptococcus thermophilus ST-4895 (DSM19242),

Streptococcus thermophilus ST6008 (also referred to as CHCC6008)(DSM18111), Lactococcus lactis, R-607-1 (also referred to as CHCC1915)(DSM21404),

Streptococcus thermophilus strain ST10255 which is commerciallyavailable from Chr. Hansen A/S as F-DVS ST-BODY-2, Material No: 623155.)

These strains are well known to the person skilled in the art, and arecommercially available from Chr. Hansen A/S.

“Fermentation” means the conversion of carbohydrates into alcohols oracids through the action of bacteria. Preferably, fermentation in themethods of the invention comprises conversion of lactose to lactic acid.Fermentation processes to be used are well known and the person of skillin the art will know how to select suitable process conditions, such astemperature, pH, oxygen, amount and characteristics of bacteria(s) andprocess time. Obviously, fermentation conditions are selected so as tosupport the achievement of the present invention, i.e. to produce (orpropagate) bacteria.

A “concentrate” as used herein is a bacteria containing culture that hasbeen concentrated, i.e. the relative number of bacteria has beenincreased by decreasing the overall volume of the culture, e.g. byremoving liquid.

LAB (Lactic Acid Bacteria), including bacteria of the speciesLactobacillus sp. and Streptococcus thermophilus, are normally suppliedto the dairy industry either as frozen or freeze-dried cultures for bulkstarter propagation or as so-called “Direct Vat Set” (DVS) cultures,intended for direct inoculation into a fermentation vessel or vat forthe production of a dairy product, such as a fermented milk product.Such cultures are in general referred to as “starter cultures” or“starters”.

LAB, including bacteria of the species Lactobacillus and Bifidobacteriumare commonly used as probiotic cultures in foods such as fermentedmilks, yoghurts and cheese, as well as dietary supplements where theprobiotic is in the form of a dried product.

A “cryoprotectant” is defined herein is a substance used to protectbacteria cells from damage during freezing, freeze-drying and thawing.The cryoprotectant may be any additive as long as it protects cells fromdamage during freezing, freeze-drying and thawing. Examples ofcryoprotectants include, but are not limited to, sugars (e.g. sucrose,fructose, trehalose), polyalcohols (e.g. glycerol, sorbitol, mannitol),polysaccharides (e.g. celluloses, starch, gums, maltodextrin),polyethers (e.g. polypropylene glycol, polyethylene glycol, polybutyleneglycol), antioxidants (e.g. natural antioxidants, such as ascorbic acid,beta-carotene, vitamin E, glutathione, or chemical antioxidants), oils(e.g. rapeseed oil, sunflower oil, olive oil), surfactants (e.g. Tween20, Tween 80, fatty acids), peptones (e.g. soy peptones, wheat peptone,whey peptone), tryptones, vitamins, minerals (e.g. iron, manganese,zinc), hydrolysates (e.g. protein hydrolysates such as whey powder, maltextract, soy), amino acids, peptides, proteins, nucleic acids,nucleotides, nucleobases (e.g. cytosine, guanine, adenine, thymine,uracil, xanthine, hypoxanthine, inosine, inositol), yeast extracts (e.g.yeast extracts of Saccharomyces spp., Kluyveromyces spp., or Torulaspp.), beef extract, growth factors, and lipids. Other examples ofcryoprotectants are disclosed in WO2012088261, WO2012076665 which areincorporated herein by reference. The addition of a cryoprotectant in aprocess of the invention may be done by mixing a solid cryoprotectantwith the bacteria concentrate for a sufficient time period at a suitabletemperature.

The term “active” (bacterial) cells as used herein refers to the numberof viable cells. The amount of active, i.e. viable, cells may bespecified in any unit or measure that is commonly used in the art. Forexample, the amount of active cells may be given in the number of viablecells per gram sample. The “total” number of (bacterial) cells as usedherein refers to the sum of active, i.e. viable, and inactive, i.e.non-viable, cells. Preferably, the total number of cells is given inabsolute terms, such as the total number of cells per gram sample. The“cell survival” as used herein refers to the percentage of active cellsrelative to the total number of cells.

Freeze-drying, also known as lyophilisation, lyophilization, orcryodesiccation, is a dehydration process typically used to preserve aperishable material or make the material more convenient fortransport/distribution. Freeze-drying works by freezing the material andthen reducing the surrounding pressure to allow the frozen water in thematerial to sublimate directly from the solid phase to the gas phase

In the present context one may use suitable freezing. As known—there areseveral herein relevant suitable freezing methods available to theskilled person, wherein some of these are fast freezing at −196° C.(liquid nitrogen) as the frozen product representing spheres orcylinders in the diameter range of 1 to 15 mm. The bacteria cells couldbe also slow frozen by placing the bacterial concentrated on producttrays or shelf trays in shelf freeze-dryers and allowing the culture tocool more slowly.

Primary drying refers to that stage where ice is transformed into vaporby sublimation.

Secondary drying refers to that stage where unfrozen water is removed bydesorption During freeze-drying, to sublimate ice (primary drying) andto desorpt bound water (secondary drying), it is necessary to supplyenergy to the sample (stuff that is going to freeze-dry). The situationfor samples on trays in the drying chamber depends on whether theproduct trays are in direct contact with the heating sources, orwheatear the product trays are suspended between the heating sourceswithout direct contact. In the first case, the sample is heated primaryby conduction from the heat sources, whereas in the second case is byradiation.

In the present context one may use suitable heating source in thefreeze-dryer. As known—there are several herein relevant suitablelarge-scale freeze-dryers available to the skilled person, wherein someof these are commercially available as with radiation heating orconduction heating. In summary, necessary energy can be transducer tothe sample in 3 different forms

-   -   By radiation of heated surface. For drying samples on trays in        the freeze-drying chamber is done through the use of radiant        heating to the sample and the tray surface.    -   By conduction from heated plates or gases. Energy transfer is by        conductivity, as well by direct contact of the product or        product container/tray with the shelfs/heating plate    -   By gas convection

Heating plates/Heating shelfs (i.e. Heating sources/surfaces): In thepresent context one may use suitable heating source in the freeze-dryer.As known—there are several herein relevant suitable large-scalefreeze-dryers availed to the skilled person, wherein some of these arecommercially available as with radiation heating or conduction heating.Heating plates as described herein may also be seen as:

-   -   heating shelfs    -   shelf heating plates    -   heating surface as shaped as product trays    -   or as any heating surface giving heat energy to ensure primary        and secondary drying.

Heating plate temperature described herein may also be seen as shelftemperature, or any temperature of the heating surface.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “having”, “including” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Theend-points of the ranges, eg. relating to the pressure, temperature orduration, can be freely combined. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

LEGENDS TO FIGURES

FIG. 1. Culture BB-12®: Comparison of unwashed and washed concentrateswith respect to a potential effect of the washing ratio on the wateractivity of freeze-dried products. The washing ratio was from 0.5 to 10volumes water per one volume concentrate. (A) washing ratio 0.5; (B)washing ratio 1; (C) washing ratio 2; (D) washing ratio 3; (E) washingratio 10; (F) No washing.

FIG. 2. Culture BB-12®: Relationship between the water activity offreeze-dried products and the washing ratio in a range of 0.5-2 volumeswater per one volume concentrate. (A) washing ratio 0.5; (B) washingratio 1; (C) washing ratio 2; (F) No washing. The line represents thelinear fit to the data with coefficient of determination R²=0.89.Equation for the linear fit: Y=−0.0485*X+0.208.

FIG. 3. Culture LGG®: Comparison of unwashed vs. washed concentrate withrespect to effect of the washing ratio on the water activity offreeze-dried products. The washing ratio was from 0.5 to 10 volumeswater per one volume concentrate. (A) washing ratio 0.5; (B) washingratio 1; (C) washing ratio 2; (D) washing ratio 3; (E) washing ratio 10;(F) No washing.

FIG. 4. Culture LGG®: Relationship between the water activity offreeze-dried products and the washing ratio in a range of 0.5-2 volumeswater per one volume concentrate (A) washing ratio 0.5; (B) washingratio 1; (C) washing ratio 2; (F) No washing. The line represents thelinear fit to the data with coefficient of determination R²=0.93.Equation for the linear fit: Y=−0.0283*X+0.166.

FIG. 5. Culture LA-5C): Comparison of water activity of freeze-driedproducts of unwashed and washed cell concentrates with respect to effectof different washing solutions on the water activity. The washing ratiowas 2, i.e. 2 volumes washing solution per one volume concentrate.Washing solutions: (A) 20% sucrose; (B) 20% trehalose; (C) phosphatebuffer pH 6; (D) water; (E) No washing.

FIG. 6. Culture LA-5®: Cell survival of frozen (pre-freeze-driedproduct, PFD) and freeze-dried (FD) products over 3 weeks; storage at30° C. and Relative Humidity of 30%. Cell survival (%) is the ratiobetween active cells and total cells. (PFD) pre-freeze-dried product;(FD) Freeze-dried product; Freeze-dried products: (Week 1) 1 weekstorage; (Week 2) 2 weeks storage; (Week 3) 3 weeks storage. The washingratio was 2, i.e. 2 volumes washing solution per one volume concentrate.Washing solutions: (A) 20% sucrose; (B) 20% trehalose; (C) phosphatebuffer pH 6; (D) water; (E) No washing.

FIG. 7. Culture LGG®: Comparison of water activity of freeze-driedproducts of unwashed (C) and washed cell concentrates with water (B) and3.4% trehalose (A) at a washing ratio of 2, i.e. 2 volumes washingsolution per one volume concentrate.

FIG. 8. Culture LGG®: Cell survival after freeze-drying and 3 weeksstorage at 30° C. and Relative Humidity of 30%. Cell survival (%) is theratio between active cells and total cells. (FD) freeze-dried product;(Week 1) 1 week storage; (Week 2) 2 weeks storage; (Week 3) 3 weeksstorage. (A) Phosphate buffer pH 6; (B) Citrate buffer pH 5; (C) 3.4%Trehalose; (D) 3.4% Sucrose; (E) 0.5% Casein peptone; (F) 0.5% Yeastextract; (G) Peptone-salt solutions; (H) 0.9% NaCl; (I) Water; (J) Nowash.

FIG. 9. Culture BB-12®: Cell survival after freezing and 3 weeks storageat 30° C. and Relative Humidity of 30%. Cell survival (%) is the ratiobetween active cells and total cells. (FD) freeze-dried product;(Week 1) 1 week storage; (Week 2) 2 weeks storage; (Week 3) 3 weekstorage. (A) Phosphate buffer pH 6; (B) Citrate buffer pH 5; (C) 3.4%Trehalose; (D) 3.4% Sucrose; (E) 0.5% Casein peptone; (F) 0.5% Yeastextract; (G) Peptone-salt solutions; (H) 0.9% NaCl; (I) Water; (J) Nowash.

FIG. 10. Culture LGG®: Comparison of the water activity of freeze-driedproducts of unwashed and washed cell concentrates with respect to aneffect of different washing solutions on the water activity. The washingratio was 2, i.e. 2 volumes washing solution per one volume concentrate.Washing solutions: (A) water; (B) 3.4% sucrose; (C) Citrate buffer pH 5(D) 3.4% trehalose; (E) No washing.

FIG. 11. Culture BB-12®: Comparison of the water activity offreeze-dried products of unwashed and washed cell concentrates withrespect to an effect of different washing solutions on the wateractivity. The washing ratio was 2, i.e. 2 volumes washing solution perone volume concentrate. Washing solutions: (A) water; (B) 3.4% sucrose;(C) Citrate buffer pH 5 (D) 3.4% trehalose; (E) No washing.

FIG. 12. Culture ST-4895: Comparison of unwashed and washed concentrateswith respect to an effect of the washing ratio on the water activity offreeze-dried products. Washing ratio from 0.5 to 10 volumes water perone volume concentrate. (A) washing ratio 0.5; (B) washing ratio 1; (C)washing ratio 2; (D) washing ratio 3; (E) washing ratio 10; (F) Nowashing.

FIG. 13. Culture ST-4895: Relationship between the water activity offreeze-dried products and the washing ratio in a range of 0.5-2 volumeswater per one volume concentrate. (A) washing ratio 0.5; (B) washingratio 1; (C) washing ratio 2; (F) No washing. The line represents thelinear fit to the data with a coefficient of determination R²=73.Equation for the linear fit: Y=−0.056*X+0.225.

FIG. 14. Culture R-607-1: Comparison of unwashed and washed concentrateswith respect to an effect of the washing ratio on the water activity offreeze-dried products. Washing ratio from 0.5 to 10 volumes water perone volume concentrate. (A) washing ratio 0.5; (B) washing ratio 1; (C)washing ratio 2; (D) washing ratio 3; (E) washing ratio 10; (F) Nowashing.

FIG. 15. Process flow diagram of the production. Scale of theproduction, washing of the cell concentrate, as well as pelletizing andfreeze-drying are shown.

FIG. 16. CultureR-607-1: Comparison of drying times between mild (A) andaggressive conditions (B-D). A (5° C., 0.3 mBar); B (50° C., 0.5 mBar);C (50° C., 0.70 mBar); D (50° C., 0.95 mBar)

FIG. 17. Culture LGG®: Comparison of drying times between mild (A) andaggressive conditions (B-D). A (5° C., 0.3 mBar); B (50° C., 0.5 mBar);C (75° C.→50° C., 0.5 mBar); D (75° C., 0.5 mBar)

EXAMPLES Example 1 Bifidobacterium animalis BB-12®: Comparison ofUnwashed Vs. Washed Concentrates with Respect to the Effect of theWashing Ratio on Water Activity of Freeze-Dried Products. Water was Usedas the Washing Solution

The Bifidobacterium animalis strain BB-12® was cultivated in MRS mediumunder standard conditions with controlled pH and temperature during thefermentation in a 700 L reactor, the culture was harvested at earlystationary phase and concentrated approx. 14 fold by centrifugation.

After centrifugation, the bacterial concentrate was divided into 6portions.

5 portions were mixed with water (deionised) in various ratios (rangingfrom 0.5 to 10) resulting in washed concentrates A-E as given below. Thewashing was done for 15 min at room temperature using a magneticstirrer. After washing, the cells were harvested by lab-scalecentrifugation (Sorvall RC 6 Plus, Thermo Scientific) for 20 minutes at10000 g and 4° C. A part of the supernatant was removed aftercentrifugation in order to obtain the original volume of theconcentrate. After that the obtained bacterial concentrate was very wellshaken in order to obtain a homogeneous bacterial suspension because thebacterial cells are in the form of a paste at the bottom of thecentrifuge tube during the centrifugation. 1 portion was used as acontrol (concentrate F).

Cryo-protective solution (which consisted of sucrose (15%), maltodextrin(10%) and water (75%)) was added (420 g to 1000 g cell concentrate) toall washed concentrates (A-E) and to the unwashed concentrate (control;F). Subsequently, all bacterial suspensions (A-F) were frozen in liquidnitrogen in a form of pellets (i.e. pre-freeze-dried product, PFD). ThePFDs were kept at −50° C. until being freeze-dried.

The following washing ratios were used:

Ratio Portion of washing solution (Liters of water per liter ofconcentrate) A 0.5 B 1 C 2 D 3 E 10 F(control) Unwashed concentrate (Noadded water)

The freeze-drying was performed in a Hetosicc freeze dryer, CD-10-1,Heto Lab equipment, Heto-Holten A/S, Allerod, Denmark as described byKurtmann. Each PFD (400 g) was put on a drying-tray. Afterwards all PFDs(A-F) were placed in the freeze-drying chamber and dried at the sametime. Immediately after freeze-drying, the water activity (a_(w)) wasmeasured for all samples. Water activity (a_(w)) of the freeze-driedmaterial was measured at room temperature using an Rotronic HYGROMER®AwVC (Rotronic Instrument Corp., Huntington, N.Y., USA). The range ofmeasurement that was achievable by use of the equipment corresponded toa water activity in the range of 0.03 to 1. Thus, the Limit of Detection(LOD) which is the lowest level that could be detected by the equipmentwas a water activity of 0.03.

The water activity of all freeze-dried products (A-F) as determinedimmediately after the freeze-drying process was compared, and theresults are presented in FIG. 1.

Results

As can been seen from FIG. 1, although all PFD products (A-F) were driedat the same time in the freeze-drying cabinet, the freeze-dried productof all washed concentrates (A-E) resulted in lower a_(w) in a range of0.03-0.12 compared to that of 0.16 for the unwashed concentrate (F).

The results also indicate that there is a threshold washing ratio abovewhich no further improvement in the water activity of freeze-driedproduct could be obtained by washing the cell concentrate. In thisexample it was found out that this threshold of the washing ratio is aratio (volume washing solution/volume concentrate) of about 2 because ahigher washing ratio than 2 did not contribute further to a lower wateractivity of the freeze-dried product.

Moreover, it was found out that a linear correlation exists between thea_(w) and the washing ratio in the range between 0 and 2 (comparesamples A, B, C, and F in FIG. 2). For example, starting from theunwashed concentrate (F) to concentrates that were washed with a ratioof 2, i.e. to the concentrates A, B and C, a_(w) linearly decreased from0.16 to 0.03. The line in FIG. 2 represents the linear fit to the data.The linearity is satisfactory with R² of 89.6%.

In conclusion, these results show that:

-   -   Washing of the cell concentrate increases the drying efficiency,        and thus washed cell concentrates of BB-12° dried better        compared to unwashed concentrate using the same drying        conditions and drying time. Thereby, washing resulted in a        significantly lower water activity (a_(w)) compared to the        sample without washing.    -   A threshold washing ratio exists. This threshold ratio is 2 in        the present experiment because a higher washing ratio than 2 did        not contribute further to a lower water activity of the        freeze-dried product.    -   A linear correlation exists between a, and the washing ratio        when the washing ratio is between 0 and 2 (i.e. 2 liters water        per liter concentrate).

Example 2 Lactobacillus rhamnosus LGG®: Comparison of Unwashed vs.Washed Concentrates with Respect to the Effect of the Washing Ratio onthe Water Activity of Freeze-Dried Products. Water was Used as theWashing Solution

The LGG® was cultivated in MRS medium under standard conditions withcontrolled pH and temperature during the fermentation in a 700 Lreactor. The culture was harvested at early stationary phase andconcentrated approx. 14 fold by centrifugation. After centrifugation,the bacterial concentrate was divided into 6 portions, washed withdeionized water, and a cryo-protective solution was added as describedin Example 1. Freeze-drying and water activity measurements wereperformed as described in Example 1.

Results

The experiment presented in this example is identical to that given inExample 1 except that it was carried out with another bacterial culture,i.e. the Lactobacillus rhamnosus LGG®. The results are presented in FIG.3.

The results of this experiment are in agreement with the findingsdescribed in Example 1, i.e. that the freeze-dried product of all washedcell concentrates (A-E) resulted in lower a_(w) compared to unwashedconcentrate (F). The water activity for washed concentrates (case A-E)was in the range of between 0.045 and 0.12 compared to 0.13 for thecontrol i.e. the unwashed concentrate (F) (FIG. 3).

These results also confirm the finding described in Example 1 that thereis a threshold washing ratio above which no further improvement of thewater activity of freeze-dried product can be obtained by washing thecell concentrate, and that this threshold washing ratio is 2.

As in Example 1, it was found that a linear correlation between a_(w)and the washing ratio exists in the washing ratio range of between 0 and2 (A, B, C, and F′; see FIG. 4). For example, starting from unwashedconcentrate (F) to to the washed concentrates (A, B and C), a_(w)linearly decreased from 0.13 to 0.045. The line in FIG. 4 represents thelinear fit to the data. The linearity is satisfactory with R² of 93.6%.

In conclusion, the results for LGG given in this experiment confirm allfindings presented in Example 1.

-   -   Washing the cell concentrates increases drying efficiency, thus        resulting in lower water activity in the washed, freeze-dried        products compared to freeze-dried products of unwashed culture        concentrates.    -   A threshold washing ratio exists. This threshold ratio is 2 in        the present experiment, because a higher washing ratio than 2        did not contribute further to a lower water activity of the        freeze-dried product.    -   A linear correlation exists between a_(w) and the washing ratio        when the washing ratio is between 0 and 2 (i.e. 2 liters water        per liter concentrate).

Example 3 Lactobacillus acidophilus LA-5®: Comparison of Freeze-DriedProducts of Washed and Unwashed Cell Concentrates with Respect to FiveDifferent Washing Solutions

The culture LA-5® was cultivated in MRS medium under standard conditionswith controlled pH and temperature during the fermentation, in a 700 Lreactor. The culture was harvested at early stationary phase andconcentrated approx. 14 fold by centrifugation.

After centrifugation, the bacterial concentrate was divided into 5portions. 4 portions were mixed with a particular washing solution (seetable below), i.e. one volume of concentrate was mixed with two volumesof washing solution as given below (A, B, C, D). 1 portion was used ascontrol (unwashed concentrate E).

The washing process, addition of cryo-protective solution,freeze-drying, and water activity measurements were performed asdescribed in Example 1. The following washing solutions were used:

Ratio of washing solution to Portion culture concentrate Washingsolution A 2 20% (w/w) Sucrose in water B 2 20% (w/w) Trehalose in waterC 2 Phosphate buffer (pH = 6) D 2 Water (deionised water) E No wash(Control)

In order to study the stability of the product with respect to cellviability during storage, freeze-dried products were stored at 30° C.and a relative humidity of 30% for 1, 2 and 3 weeks. Subsequently,samples were taken out for Flow cytometry analysis as described in WO2006/125446.

The flow cytometry method was used to quantify active versus inactivecells on the level of energy metabolism. Active cells, i.e. the numberof viable cells per gram sample, are detected by a cellular fluorescencestaining, which differentiates cells generating a membrane potentialfrom cells without such functional energy metabolism as described. Inthe text:

-   -   Active cells refer to number of viable cells per gram sample    -   Total cells refer to sum of active and inactive cells    -   Cell survival refers to % of active cells relative to total        cells

Results 3.1: Comparison of the Water Activity of Freeze-Dried Productsof Washed and Unwashed Cell Concentrates with Respect to DifferentWashing Solutions

The aim of this example was to study the effect of different washingsolutions on the water activity of washed freeze-dried products comparedto that of unwashed freeze-dried products. In this example, a cellconcentrate of a culture of LA-5® was used. A washing ratio of 2 (i.e. 2volumes washing solution per volume concentrate) was selected in orderto provide more profound data on potential synergetic effects of thewashing ratio and the type of washing solution.

The water activities of all freeze-dried (FD) products (A-E), asdetermined immediately after the freeze-drying process were compared.The results are presented in FIG. 5. Although different washingsolutions were used, all washed FD products (A-D) resulted in a lowera_(w) of 0.03-0.04 as compared to that of 0.08 of unwashed FD product(case E). All PFD products were dried at the same time in thefreeze-dryer.

Moreover, the cell concentrate washed with water (D) resulted in a FDproduct with an a_(w) of 0.03 which is comparable to the water activity(0.03-0.04) of freeze-dried products washed with sucrose solution (A),trehalose solution (B) or phosphate buffer (C). This demonstrates thatthe major effect on obtaining a low a, for the FD product comes from thewashing process itself and not from the particular washing solutionused.

Results 3.2: Cell Survival in Washed and Unwashed Cell Concentrates ofStrain LA-5® During Storage

Results for cell survival, i.e. the % of active relative to total(active plus inactive) cells, of all FD products of LA-5® afterfreeze-drying and 1, 2 and 3 weeks storage at 30° C. and a relativehumidity of 30% are summarized in FIG. 6. The cell survival of all FDproducts after freeze-drying is near 90%. A significant drop in cellsurvival of about 31-35% occurred after 1 week storage for FD productswashed with phosphate buffer (C) and with water (D) as well as forunwashed FD product (E). The lowest cell survival of 24% after 3 weeksof storage was obtained for the unwashed FD product (case E), for whichcell survival was 40% and 51% lower compared with the highest cellsurvival obtained by washing the cell concentrate with sucrose solution(B) or trehalose solution (A).

These results confirm the findings in Example 5 demonstrating thatwashing of the cell concentrate results in a higher survival duringlong-term storage, and that some washing solutions like sucrose andtrehalose are more efficient as compared to others with respect topreserving the cell viability during washing and subsequentfreeze-drying and storage.

Overall, the conclusions that can be drawn from the results presented inExample 3 with cultures of the strain LA-5® are:

-   -   Washing of the cell concentrate increases the drying efficiency,        thus resulting in a lower water activity in washed freeze-dried        products compared to freeze-dried products of unwashed culture        concentrate. This finding is in agreement with the results given        in Examples 1 and 2.    -   The low water activity of washed FD products compared to        unwashed FD products is due to the effect of the washing process        itself, and is not attributable to the effect of a particular        washing solution used.    -   Compared to the unwashed FD product, the washing with some        particular solutions such as sucrose and trehalose results not        only in a lower water activity of the FD products but also in        more stable FD products with respect to preserving cell        viability during storage.

Example 4 Lactobacillus rhamnosus LGG®: Comparison of the Water Activityof Freeze-Dried Products of Washed and Unwashed Cell Concentrates withRespect to Different Washing Solutions, i.e. Water and TrehaloseSolution

The Lactobacillus rhamnosus strain LGG® was cultivated in 800 liters MRSmedium under standard conditions with controlled pH and temperature. Theculture was harvested at early stationary phase and concentrated approx.13 fold by centrifugation.

The bacteria-containing concentrate was divided into 3 portions, eachcomprising a volume of 20 liters. For two of the portions, thebacteria-containing culture concentrate (20 liters) was pumped into atank, and 40 liters of deionized water (A) or 3.4% trehalose (B) wereadded. The resulting suspension was agitated, and concentrated to 20liters using a centrifuge (CSA-6, Westfalia).

The following washing solutions were used:

Ratio of washing solution to Portion culture concentrate Washingsolution A 2 Water (deionised water) B 2 3.4% (w/w) Trehalose in water C2 No wash (Control)

After that, 10 liters of each washed concentrate (A and B) were mixedwith cryo-protective solution as described in Example 1 above. Oneportion was used as a control (concentrate C).

The addition of cryo-protective solution was performed as described inExample 1. Afterwards all bacterial suspensions (A-C) were frozen inliquid nitrogen in the form of pellets (i.e. pre-freeze-dried products,PFD). The PFDs were kept at −50° C. until being freeze-dried.

The freeze-drying was performed as described in Example 1 with thefollowing modifications: In total three freeze-dryings were carried out,i.e. one for each PFD bulk, as described previously by Kurtmann L et al.(2009), Biotechnol Prog. 25(1):265-70. Each freeze-drying was carriedout for 22 hours with a PFD load in the drying cabinet of 10 kg. Thewater activity of each FD product was measured immediately afterfreeze-drying.

The water activity measurement was performed as described in Example 1.

Results

The aim of this example was to study the effect of two different washingsolutions on the water activity of washed freeze-dried products comparedto that of unwashed freeze-dried product. In this example, the cellconcentrate of a culture of strain LGG® was used. A washing ratio of 2was chosen in order to provide more profound data on potentialsynergetic effects of the washing ratio and the washing solution.

The water activity of all freeze-dried (FD) products (A-C), asdetermined immediately after the freeze-drying process was compared. Theresults are presented in FIG. 7. The FD product washed with trehalosesolution (B) had a similar a, (0.027) compared to that of the FD productwashed with water (a, =0.022; A). Thus, both washed FD products (A andB) resulted in an a, below the Limit of Detection (LOD) of 0.03 for theequipment indicating that the samples were much more dried compared tothe unwashed FD product (a_(w)=0.08; C), although they had beenfreeze-dried for the same duration. In conclusion, these results are inagreement with the conclusions made in Examples 1; 2; and 3 above whichfound that:

-   -   Washing of a cell concentrate results in a lower water activity        in FD products as compared to unwashed FD product when the FD        products are freeze-dried under the same drying conditions and        for the same time.    -   The low water activity of washed FD products compared to        unwashed FD products is due to the washing process itself, and        is not attributable to the effect of a particular washing        solution used.

Example 5 Cultures BB12 and LGG®: Cells Washed with Nine DifferentWashing Solutions

The experiment was performed for cultures of the strains BB-12® andLGG®. The bacteria-containing concentrate of BB-12® and LGG® was made asdescribed in Examples 1 and 2, respectively. After centrifugation, onevolume of concentrate BB-12® or LGG® was mixed with two volumes washingsolution. In total, 9 different washing solutions were used as describedbelow (A-I, Table 1). One portion was used as a control (concentrate J).

The washing process, addition of cryo-protective solution, andfreeze-drying were performed as described in Example 1.

The stability of freeze-dried formulations (A-J) was then evaluated withrespect to the cell viability during storage for 1, 2 and 3 weeks at 30°C. with a relative humidity of 30%. After that, samples were taken outfor Flow cytometry analysis as described in Example 3.

TABLE 1 Washing solutions Portion Solutions pH A Phosphate buffer 6.1 BCitrate buffer 5.04 C 3.4% Trehalose 6.54 D 3.4% Sucrose 6.43 E 0.5%Casein peptone 6.78 F 0.5% Yeast Extract 5.86 G Peptone Saline Diluent6.9 H NaCl 6.37 I Water (deionised water) 6.10 J No wash

Results

The aim of this example was to study the effect of different washingsolutions with respect to preserving cell viability (cell survival)during freeze-drying and storage at 30° C. with a relative humidity of30%. A washing ratio of 2 was chosen in order to provide more profounddata on potential synergetic effects of the washing ratio and thewashing solution. Nine different washing solutions, including water,were tested as shown in Table 1. The results of cell survival offreeze-dried product after freeze-drying (FD) and after 1, 2 and 3 weeksstorage at 30° C. with a relative humidity of 30% are presented in FIG.8 for LGG® and FIG. 9 for BB12.

For LGG®, a decrease in cell survival was already seen after storage for1 week. The most significant cell loss of 33% occurred with the unwashedconcentrate (J), followed by 20% cell loss with concentrate washed withNaCl (0.9%) (H). For all FD products (A-J), the cell survival after 2weeks of storage was similar to that after 3 weeks. The lowest cellsurvival of 17% after 3 weeks of storage was obtained with the unwashedconcentrate (J), which was 31% lower compared with the highest cellsurvival obtained by washing the concentrate with peptone salinesolution (48%) (G). The second highest cell survival of 41% was obtainedby washing with trehalose solution (C) which was still significantlyhigher, i.e. 21%, in comparison with the unwashed FD product (J).Washing with water (I) also resulted in a 11% higher cell survival after3 weeks storage compared to the unwashed FD product (J).

For BB-12®, as shown in FIG. 9, the cell survival for all FD productsafter freeze-drying (A-J) was about 90%. Cell survival after 3 weeks ofstorage was in the range of 80-85%. The lowest cell survival of 62-63%after 3 weeks of storage was obtained with the unwashed concentrate (J),and this survival was in the range of 10-23% lower compared to the otherwashed FD products (A-I).

In summary, the results from these experiments with both cultures ofLGG® and BB-12® demonstrate that washing of the cell concentrate resultsin higher cell survival during long-term storage. Moreover, thedifference seen in cell survival after 3 weeks storage also indicatesthat certain washing solutions are more effective than others withrespect to preserving cell viability during washing and subsequentfreeze-drying and storage.

Example 6 Lactobacillus Rhamnosus LGG®: Comparison of the Water Activityof Freeze-Dried Products of Washed and Unwashed Cell Concentrates withRespect to 4 Different Washing Solutions

The culture LGG® was cultivated in MRS medium under standard conditionswith controlled pH and temperature during fermentation, in a 700 Lreactor. The culture was harvested at early stationary phase andconcentrated approx. 14 fold by centrifugation.

After centrifugation, the bacterial concentrate was divided into 5portions. 4 portions were mixed with a particular washing solution (seetable below). In particular, one volume of concentrate was mixed withtwo volumes of washing solution as shown in the Table below (A, B, C,D). One portion was used as a control (unwashed concentrate E).

The washing process, addition of cryo-protective solution,freeze-drying, and water activity measurement was performed as describedin Example 1.

The following washing solutions were used

Ratio of washing solution to Portion culture concentrate Washingsolution A 2 Water (deionised water) B 2 3.4% (w/w) sucrose in water C 2Citrate buffer (pH = 5) D 2 3.4% (w/w) trehalose in water E No wash(Control)

Results

The aim of Example 6 was to study the effect of 4 different washingsolutions on the water activity of washed freeze-dried products comparedto that of unwashed freeze-dried product. In this example, a cellconcentrate of a culture of the strain LGG® was used. A washing ratio of2 (2 volumes washing solution per volume concentrate) was chosen inorder to provide more profound data on potential synergetic effects ofthe washing ratio and the washing solution.

The water activity of all freeze-dried (FD) products (A-E), asdetermined immediately after the freeze-drying process was compared. Theresults are presented in FIG. 10. Although different washing solutionswere used, all washed FD products (A-D) resulted in very low a_(w) ofless than 0.03 compared to that of 0.072 of unwashed FD product (caseE). All PFD products were dried at the same time in the freeze-dryer. Inaddition, all washed FD products (A-D) resulted in a_(w) below the Limitof Detection (LOD) of 0.03 for the equipment indicating that the sampleswere more dried compared to the unwashed sample (E). This demonstratesthat the strong effect on obtaining a low a, for the FD product comesfrom the washing process itself and is not attributable to theparticular washing solution used.

In conclusion, these results are in agreement with the conclusions madein Examples 1; 2; 3; 4 and 7 which found that:

-   -   Washing of the cell concentrate results in a lower water        activity in FD products as compared to unwashed FD product when        both have been freeze-dried under the same drying conditions and        duration.    -   A low water activity of washed FD products compared to that of        unwashed FD products is due to the effect of the washing process        itself, and not attributable to the effect of a particular        washing solution that is used.

Example 7 Bifidobacterium Culture, BB-12®: Comparison of the WaterActivity of Freeze-Dried Products of Washed and Unwashed CellConcentrates with Respect to 4 Different Washing Solutions

A culture of the strain BB-12® was cultivated in MRS medium understandard conditions with controlled pH and temperature during thefermentation, in a 700 L reactor. The culture was harvested at earlystationary phase and concentrated approx. 14 fold by centrifugation.

After centrifugation, the bacterial concentrate was divided into 5portions. 4 portions were mixed with a particular washing solution (seetable below). In particular, one volume of concentrate was mixed withtwo volumes of washing solution as given below (A, B, C, D). One portionwas used as a control (unwashed concentrate E).

The washing process, addition of cryo-protective solution,freeze-drying, and water activity measurement were performed asdescribed in Example 1.

The following washing solutions were used

Ratio of washing solution to Portion culture concentrate Washingsolution A 2 Water (deionised water) B 2 3.4% (w/w) sucrose in water C 2Citrate buffer (pH = 5) D 2 3.4% (w/w) trehalose in water E No wash(Control)

Results

The aim of example 7 was to study the effect of different washingsolutions on the water activity of washed freeze-dried products comparedto that of unwashed freeze-dried product. Thus, a washing ratio of 2(i.e. 2 volumes washing solution per volume concentrate) was chosen inorder to provide more profound data on potential synergetic effects ofthe washing ratio and the washing solution. The experiment presented inthis example was identical to that described in Example 6 except that itwas carried out with another culture of BB-12®.

The water activity of all freeze-dried (FD) products (A-E), asdetermined immediately after the freeze-drying process was compared, andthe results are presented in FIG. 11. Although different washingsolutions were used, all washed FD products (A-D) resulted in a lowera_(w) in the range of 0.013-0.021 as compared to that of 0.047 ofunwashed FD product (E). All PFD products were dried at the same time inthe freeze-dryer. In addition, all washed FD products (A-D) resulted inan a_(w) below the Limit of Detection (LOD) of 0.03 for the equipmentindicating that the samples were more dried compared to the unwashedsample (E). This also demonstrates that the main effect on obtaining alow a_(w) for FD product comes from the washing process itself and notthe particular washing solution used.

In conclusion, these results are in agreement with the conclusions madein Examples 1; 2; 3; 4 and 6 which described that:

-   -   Washing of cell concentrates results in a lower water activity        in the FD product compared to unwashed FD product when both have        been freeze-dried under the same drying conditions and time.    -   The low water activity of washed FD products compared to        unwashed FD products is due to the effect of washing process        itself, and not attributable to the effect of the particular        washing solution used.

Example 8 Streptococcus thermophilus, ST-4895: Comparison of Unwashedand Washed Concentrates with Respect to Effect of Washing Ratio on WaterActivity of Freeze-Dried Products. Phosphate Buffer (pH 6) was Used asthe Washing Solution

The strain ST-4895 was cultivated in MRS medium under standardconditions with controlled 25 pH and temperature during the fermentationin a 700 L reactor. The culture was harvested at early stationary phaseand concentrated approx. 14 fold by centrifugation. Aftercentrifugation, the bacterial concentrate was divided in 6 portions.Five portions (A-E) were washed with phosphate buffer (pH 6) and 1portion was used as a control (unwashed concentrate F) as described inExample 1.

Addition of cryo-protective solution was performed as described inExample 1.

Freeze-drying was performed as described in Example 1 with the followingmodification: a pressure of 0.5 mBar was used instead of 0.3 mBar. Wateractivity measurement was performed as described in Example 1.

Results

The experiment presented in this example was identical to that describedin Examples 1, 2 and 9 except that it was carried out with anotherculture, i.e. a culture of the strain ST-4895, and that phosphate bufferwith pH 6 was used as a washing solution. The results are presented inFIGS. 12 and 13.

Although a different washing solution, i.e. phosphate buffer at pH 6,was used compared to water used in Examples 1 and 2, and the peptonesaline diluent used in Example 9, the results in this example are inagreement with the findings in the above Examples (1, 2 and 9).Specifically, the freeze-dried product of all washed cell concentrates(A-E) had a lower a_(w) as compared to the unwashed concentrate (F). AllPFD products were dried at the same time in the freeze-dryer. The wateractivity of the washed concentrates (A-E) was in the range of 0.03-0.6compared to 0.22 for the control, i.e. the unwashed concentrate (F)(FIG. 12).

Although phosphate buffer pH 6 was used as a washing solution in thisexample, all washed FD products (A-E) had a very low a_(w) (0.03-0.06)which confirms the above results obtained with freeze-dried productswashed with water (A-E) in Examples 1 and 2; and with peptone salinediluent in Example 9. These results are also in agreement with thefindings presented in examples 3, 4 6 and 7 which showed that the maineffect on obtaining a low a_(w) in FD products comes from the washingprocess itself and not from the particular washing solution used.

Although phosphate buffer pH 6 was used as a washing solution in thisexample, the results also confirm the findings in Examples 1, 2 and 9that there is a threshold washing ratio above which no furtherimprovement in water activity of freeze-dried product will be obtainedby washing the cell concentrate, and that this threshold washing ratiois 2. This demonstrates that the threshold washing ratio of 2 does notdepend on the particular washing solution used, but it is due to thewashing process itself.

As in Examples 1, 2 and 9, it was found out that a linear correlationexists between a_(w) and the washing ratio in the range of 0 to 2 (A, B,C, and F) (FIG. 13). For example, starting from the unwashed concentrate(F) to concentrate that was washed with a washing ratio of 2 (A, B andC), the a_(w) linearly decreased from 0.22 to 0.03. The line in FIG. 13represents the linear fit to the data. Linearity is satisfactory with R²of 73.6.

In conclusion, the results for ST-4895 described in this experimentconfirm all findings presented in Examples 1; 2 and 9.

-   -   Washing the cell concentrate increases the drying efficiency,        thus resulting in a lower water activity in washed freeze-dried        products compared to freeze-dried product of unwashed culture        concentrate.    -   A threshold washing ratio exits and is 2 in this experiment; A        higher washing ratio than 2 does not contribute further to        decreasing the water activity of freeze-dried product.    -   The threshold washing ratio of 2 does not depend on the washing        solution used, but it is due to the washing process itself.    -   A linear correlation exists between a_(w) and the washing ratio        as starting from unwashed to concentrates that were washed with        a washing ratio of 2 (i.e. 2 liters water per liter        concentrate).    -   The low water activity of washed FD products compared to        unwashed FD products is due to an effect of the washing process        itself, and not to the effect of a particular washing solution        used, which is in agreement with the findings presented in        examples 3, 4, 6 and 7.

Example 9 Lactococcus Lactis, R-607-1: Comparison of Unwashed and WashedConcentrates with Respect to Effects of the Washing Ratio on the WaterActivity of Freeze-Dried Products. Peptone Saline Diluent (pH 6.9) wasUsed as the Washing Solution

The strain R-607-1 was cultivated in MRS medium under standardconditions with controlled 15 pH and temperature during the fermentationin a 700 L reactor. The culture was harvested at early stationary phaseand concentrated approx. 14 fold by centrifugation. Aftercentrifugation, the bacterial concentrate was divided into 6 portions.Five portions (A-E) were washed with peptone saline diluent (pH 6.9) and1 portion was used as a control (unwashed concentrate F) as described inExample 1.

Addition of cryo-protective solution was performed as described inExample 1.

Freeze-drying was performed as described in Example 1 with the followingmodification: a pressure of 0.5 mBar was used instead of 0.3 mBar. Thewater activity measurement was performed as described in Example 1.

Results

The experiment presented in this example was identical to that describedin Examples 1, 2 and 8 except that it was carried out with anotherculture, i.e. a culture of the strain R-607-1, and that peptone salinediluent (pH 6.9) was used as washing solution. The results are presentedin FIG. 14.

Although a different washing solution, i.e. peptone saline diluent (pH6.9), was used (and not water as in Examples 1 and 2, or phosphatebuffer at pH 6 as in Example 8), the results in this example are inagreement with the findings in the above Examples (1, 2 and 8), i.e. thefreeze-dried product of all washed cell concentrates (A-E) had a lowera_(w) compared to unwashed concentrate (F). All PFD products were driedat the same time in the freeze-dryer. Water activity of washedconcentrates (A-E) was equal or below 0.03 compared to an a_(w) of 0.08of the control, i.e. the unwashed concentrate (F) (FIG. 14). Inaddition, washed FD products (B-D) had an a_(w) below the Limit ofDetection (LOD) of 0.03 for the equipment indicating that the sampleswere more dried compared to the unwashed sample (F). This also confirmsthe findings presented in examples 3, 4 6 and 7 that the main effect onobtaining a low a_(w) for FD product results from the washing processitself and not from the particular washing solution used.

Although peptone saline diluent (pH 6.9) were used as washing solutionin this example, the results also confirm the findings in Examples 1, 2and 8 that there is a threshold washing ratio above which no furtherimprovement in water activity of the freeze-dried product will beobtained by washing the cell concentrate, and that this thresholdwashing ratio is 2. This demonstrates that the threshold washing ratioof 2 does not depend on the washing solution used, but is due to thewashing process itself.

In conclusion, the results for R-607-1 given in this example confirm allfindings presented in Examples 1; 2 and 8.

-   -   Washing of the cell concentrates increases the drying        efficiency, thus resulting in a lower water activity in washed        freeze-dried products compared with dried product of unwashed        culture concentrate.    -   A threshold washing ratio exits and is 2; as higher washing        ratio than 2 does not contribute further to a lower water        activity of a freeze-dried product.    -   The threshold washing ratio of 2 does not depend on the washing        solution used, but it is due to the washing process itself.    -   The low water activity of washed FD products compared to        unwashed FD products is due to the effect of the washing process        itself, and not to the effect of a particular washing solution        used in agreement with the findings of Examples 3, 4, 6 and 7.

Example 10 Lactobacillus rhamnosus LGG®: Comparison of ProductAppearance and Water Activity of Freeze-Dried Products of Washed andUnwashed Products when Freeze-Dried at Different Pressure

The strain LGG® was cultivated in 800 liters MRS medium under standardconditions with controlled pH and temperature. The culture was harvestedat early stationary phase and concentrated approx. 13 fold bycentrifugation.

The concentrated culture was divided into 2 portions of 20 liters each.One of the portions of the culture concentrate (20 liters) was pumpedinto a tank, and 40 liters of deionized water (A) were added. Theresulting suspension was agitated and concentrated to 20 liters using acentrifuge (CSA-6, Westfalia) resulting in a washed concentrate (A). Thesecond portion was used as control i.e. unwashed concentrate(concentrate B).

The addition of cryo-protective solution was performed as described inExample 1. Afterwards both bacterial suspensions (A-B) were frozen byliquid nitrogen in the form of pellets (i.e. PFD). The PFDs were kept at−50° C. until being freeze-dried.

The following washing solutions were used

Ratio of washing solution to Portion culture concentrate Washingsolution A 2 Water (deionised water) B 2 No wash (Control)

The freeze-drying was performed as described in Example 1 with thefollowing modifications. Pre-freeze-dried product (PFD) was dried atfive different chamber pressures (0.3; 0.5; 0.7; 0.9 and 1.2 mBar). Oneparticular chamber pressure was used per freeze-drying, so in total fivefreeze-dryings were done per PFD product (A and B). 4000 g PFD were usedper drying.

Water activity measurement (a_(w)) of each FD product (A and B) wasperformed immediately after freeze-drying as described in Example 1.

After freeze-drying under various chamber pressures, the freeze-driedproducts were visually assessed for product appearance, which was doneby a visual comparison to a reference. As a reference, a freeze-driedproduct of unwashed concentrate (portion B) which was freeze-dried at apressure of 0.3 mBar was used.

The following categorization was used for evaluation of freeze-driedproduct appearance after freeze-drying:

-   -   0 refers to products similar to the reference, i.e. normal        pellet shape    -   1 refers to product with a less normal pellet shape    -   2 refers to product with bad pellet shape    -   3 refers to product with complete loss of pellet shape

Results Evaluation of freeze-dried products normally includes theobservation of the product appearance after freeze-drying. One of thedesired characteristics of freeze-dried products includes avoidingchanges in the fried-product product appearance. Thus, the aim of thisexample was to study the effect of washing on product appearance whenproducts undergo freeze-drying at various pressures. Two types ofpre-freeze-dried products (PFDs: A-B) were used. The first PFD (A) waswashed with water and the second one (B) was the control, i.e. unwashedPFD. Five freeze-dryings were performed per PFD product at a constantshelf temperature of 32° C., and the pressure was increased graduallyfrom 0.3 to 1.2 mBar. The product appearance after freeze-drying underdifferent pressures was assessed by visual comparison to a referencefreeze-dried product which was unwashed concentrate (portion B) andfreeze-dried at a pressure of 0.3 mBar. The results for productappearance after freeze-drying and water activity of freeze-driedproducts are shown in Table 2 and 3, respectively.

At the lowest pressure of 0.3 mBar, no difference in products appearancewas observed for the two products (A and B), although unwashed FDproduct (B) had a higher a_(w) of 0.08 (Table 2). For the unwashedproduct (B), increasing gradually the pressure from 0.5 to 1.2 mBarresulted in a gradual loss of pellet shape corresponding to a degree ofappearance from 1 to 3 (Table 2). The bad product appearance atpressures in the range of 0.5-1.2 mBar was also confirmed by the highvalues of water activity (a_(w)) in the range of 0.21-0.35 (Table 3).

The product washed with water (A) was successfully freeze-dried at alltested pressures in the range of 0.3 to 1.2 mBar, and no visual changein product appearance (i.e. degree of 0) was seen (Table 2). The factthat product appearance and characteristics remained unchanged at allpressures tested was also confirmed by the very low value of the wateractivity (a_(w)) of 0.03, which was below the Limit of Detection (LOD)of 0.03 for the equipment (Table 3). This indicates that the product wasvery well dried (Table 3).

TABLE 2 Culture LGG ®: Comparison of product appearance of freeze-driedproducts of unwashed and washed cell concentrates with respect to apotential effect of the pressure (0.3 to 1.2 mBar) during freeze-drying.The washing ratio is 2, i.e. 2 volumes washing solution (water) per onevolume concentrate. Washing solutions: (A) water; (B) No washing.Pressure during drying 0.3 mBar 0.5 mBar 0.7 mBar 0.9 mBar 1.2 mBarPortion Evaluation of product appearance after freeze-drying A 0 0 0 0 0(washed with water) B 0 0 1 2 3 (No wash)

TABLE 3 Culture LGG ®: Comparison of water activity of freeze-driedproducts of unwashed and washed cell concentrates with respect to apotential effect of the pressure (0.3 to 1.2 mBar) during freeze-drying.The washing ratio is 2, i.e. 2 volumes washing solution (water) per onevolume concentrate. Washing solutions: (A) water; (B) No washing.Pressure during drying 0.3 mBar 0.5 mBar 0.7 mBar 0.9 mBar 1.2 mBarPortion water activity (a_(w)) of freeze-dried products A <0.03 <0.03<0.03 <0.03 <0.03 (washed with water) B 0.08 0.213 0.32 0.31 0.35 (Nowash)

In conclusion, these results show that:

-   -   Washing of the cell concentrates reduces the risks of changes in        product appearance during freeze-drying, especially at high        pressure.    -   Washing of the cell concentrates with water allows drying at        higher pressure of 1.2 mBar compared to the unwashed        freeze-dried products with no change in visual product        appearance. This was also confirmed by the very low value of        water activity (a_(w)) of 0.03, which is below the Limit of        Detection (LOD) for the equipment.    -   Even at a higher pressure of 1.2 mBar, washed freeze-dried        product had the same low water activity (a_(w)) of less than        0.03 compared to a pressure of 0.3 mBar, which is below the        Limit of Detection (LOD) of 0.03 for the equipment    -   The findings in this example are in agreement with the results        given in Examples 1, 2, 3, 4, 6, 7, 8 and 9; i.e. the washing of        cell concentrates improves the drying efficiency by resulting in        lower water activity compared to the unwashed freeze-dried        products, even at higher pressure.

Example 11 Hardness of Washed Freeze-Dried Products

The experiment presented in this example was identical to that describedin Example 2.

Five washed freeze-dried products were prepared (A-E) using a differentwashing ratio in the range of 0.5 to 10 (liters of water per liter ofconcentrate) as described in Example 2. Product (F) was the control,i.e. the unwashed freeze-dried product.

The aim of this example was to evaluate the effect of washing on thehardness of the freeze-dried pellets (Table 4). The hardness of thefreeze-dried pellets was evident visually and by feeling after pressingthe pellets physically by hand (Table 4). Washing the cell concentratereduced the concentration of unused media components and fermentationproducts which resulted in less hard freeze-dried pellets (A-E), whichcan be easily broken compared to the unwashed (F) product. The washedproducts (A-E) also look and feel fluffier and most likely have a moreporous. With regard to how hard it is to break the freeze-driedproducts, the pellet hardness of washed products (A-E) decreased fromwashing ratio 0.5 to 2 and a washing ratio of 2 resulted in similarlybrittle freeze-dried pellets as washing ratios of 3 and 10. Based onthese results it is expected that the washed freeze-dried product can begrinded more easily. Thus, less heat will be developed during grinding,the grinding step will be shortened and, consequently, better survivalduring grinding will be observed.

TABLE 4 Culture LGG ®: Evaluation of the effect of washing onfreeze-dried pellet hardness with respect to how hard it is to break thefreeze-dried products. Washing ratio from 0.5 to 10 volumes water perone volume concentrate. (A) washing ratio 0.5; (B) washing ratio 1; (C)washing ratio 2; (D) washing ratio 3; (E) washing ratio 10; (F) Nowashing. Ratio of washing solution (Liters of water per liter Portion ofconcentrate) Appearance A 0.5 Very crisp pellets, yellow, rough surface,small pellets B 1 Crisp pellets, light yellow, rough surface, easy togrind C 2 Brittle pellets, light yellow, rough surface, easy to grind D3 Brittle pellets, light yellow, rough surface, easy to grind E 10Brittle pellets, white, rough surface, easy to grind F (control)Unwashed Very hard, sticky pellets, dark yellow, concentrate (No roughsurface, small pellets added water)

Overall the results from this study indicate that:

-   -   Washing the cell concentrate with water decreases the pellet        hardness resulting in pellets that can be broken easily compared        to the unwashed freeze-dried product.    -   With regard to how hard is to break the freeze-dried products,        the pellet hardness of washed products decreased with increasing        washing ratio from 0.5 to 2    -   With regard to how hard it is to break the freeze-dried        products, the washing ratio of 2 resulted in similarly brittle        freeze-dried pellets as the washing ratios 3 and 10.

Example 12 Wash of a Lactobacillus acidophilus, LA-5®

The strain La-5® is cultivated in 7000 liters MRS medium under standardconditions with controlled pH and temperature.

The culture is harvested at early stationary phase and concentrated14-fold by centrifugation, using a continuous centrifuge Alfa-Laval MRPX418 SGV-34C.

The concentrated culture (500 liters) is pumped into a tank, and 1000liters of deionized water are added. The resulting suspension isagitated and concentrated to 500 liters using a continuous centrifugeAlfa-Laval MRPX 418 SGV-34C. The production process flow is shown inFIG. 15.

The addition of cryoprotective solution is performed as described inExample 1. The concentrated culture is freeze-dried.

Example 13 Wash of a Bifidobacterium Culture, BB-12®

The strain BB-12 is cultivated in 7000 liters MRS medium under standardconditions with controlled pH and temperature. The culture is harvestedat early stationary phase and concentrated 14 fold by centrifugation,using a continuous centrifuge (Alfa-Laval MRPX 418 SGV-34C).

The concentrated culture (500 liters) is pumped into a tank, and 1000liters of deionized water are added. The resulting suspension isagitated and concentrated to 50 liters using a continuous centrifuge.The production process flow is shown in FIG. 15.

The addition of cryo-protective solution is performed as described inExample 1. The concentrated culture is freeze-dried.

Example 14 Lactococcus Lactis, R-607-1: Comparison of Product Qualitywhen Freeze-Dried Under Mild and Aggressive Conditions. Effect of HighPressure (0.5-0.95 mBar)

Lactococcus lactis, R-607-1 ® with deposit accession number DSM21404 wascultivated in 700 liters MRS. The bacteria in the fermentation brothwere concentrated by centrifugation using a centrifuge CSA-6 Westfalia.After centrifugation, the bacterial concentrate was mixed withcryoprotective solution (300 g to 1000 g cell concentrate). Afterwardsthe bacterial suspension was frozen with liquid nitrogen in the form ofpellets (i.e. PFD). The cryoprotective solution consisted of skim milk(14%), monosodium glutamate (6%) and water (80%). The frozen pellets ofthe bacterial suspension are called pre-freeze-dried product (i.e. PFD).Pre-freeze-dried product (PFD) in the form of frozen pellets (i.e. PFD)with a size of 1 to 5 mm in diameter was used for the freeze-dryingtrials carried out in the pilot scale described below. The PFD was keptat −50° C. until being freeze-dried.

The freeze-drying was performed in a Hetosicc freeze dryer, CD-10-1,Heto Lab equipment, Heto-Holten A/S, Allerod, Denmark.

The freeze-dryer can operate at pressures in the range of 0.2-2 mBar andis equipped with a heating plates that operates in the range of −40 to+80° C. The condenser operates with an average temperature of −60° C.The amount of material required is between 0 and 10 kg. The freeze-dryerhas 6 heating plates designed for Radiant drying and a supporting rackfor the trays. The supporting rack for the trays is suspended in aweighing cell (see Atlas Pilot Freeze-drying Plant-RAY™, NIRO, DK). Theweighing device is connected to a computer which allows the recording ofthe mass, i.e. the change in weight during drying due to removal ofwater, thereby assuring an accurate process control. The drying traysare located between the heating plates by hanging on the rack.Therefore, a maximum of 5 levels of trays can be positioned as thenumber of heating plates is 6. In this study, 2 trays made of anodisedaluminium 470×250×35 mm, were positioned per level, so that in total 10trays were present in the freeze-drying cabinet (2 trays per level×5levels=10 trays).

Frozen pellets (PFD) of R-607-1 with a mass of 10 kg were put on thedrying trays and afterwards placed in the freeze-drying chamber anddried with 4 different freeze-drying (FD) cycles as indicated below.Freeze-drying (FD) cycle A was a control cycle, i.e. a so-called mildcycle, with a constant heating plate temperature and a constant chamberpressure during the drying of 5° C. and 0.3 mBar, respectively. Theother freeze-drying cycles (B, C and D) were carried out using moreaggressive drying conditions. A higher pressure of 0.5, 0.7 and 0.95mBar was applied for FD cycle B, C and D, respectively. The heatingplate temperature was also increased from 5 to 50° C. Both pressure andheating plate temperature were kept constant during the drying process(B, C and D).

The end of the drying was reached when stable weight during the dryingwas reached, and the product temperature was not higher than 35° C.

The following freeze-drying cycles were used:

Pressure Heating plate temperature Freeze-drying cycle mBar ° C. A(Control) 0.3  5° C. B 0.5 50° C. C 0.7 50° C. D 0.95 50° C.

The water activity (a_(w)) of freeze-dried products was measuredimmediately after freeze-drying. Water activity (a_(w)) measurement wasperformed as described in Example 1.

The acidification activity in the freeze-dried culture was measuredaccording to the International standard ISO 26323:2009 (IDF 213: 2009):“Milk products—Determination of the acidification activity of dairycultures by continuous pH measurement (CpH)”.

Acidification activity is qualified by the following parameters:

-   -   t_(a): The time it takes to start acidifying the standardized        milk, i.e. the time in which the pH drops 0.08 pH units from the        initial pH. The time t_(a) is measured in minutes from the time        of inoculation, t=0.    -   pH-6h: The pH that is reached after 6 hours at 30° C. for this        particular starter culture.    -   The higher t_(a) and pH-6h are, the longer the latency phase        and, thus, the lower the acidification activity (Fernanda et al.        2004).

The evaluation of the appearance of the freeze-dried product afterfreeze-drying and the categorization was performed according to thegrading described in Example 10.

Results

The aim of the present study was to investigate the effect of aggressivefreeze-drying conditions (high pressure) on the quality of freeze-driedproducts of R-607-1. Examination was done by comparison of both wateractivity (a_(w)) and acidification activity (t_(a), pH-6h) offreeze-dried products obtained by aggressive (B-D) and mild (A) FDcycles (see table above). The mild drying (A, reference drying) wascarried out with a constant heating plate temperature of 5° C. and witha constant chamber pressure of 0.3 mBar. For the aggressive dryings(B-D), the pressure was increased from 0.3 mBar (A) to 0.5, 0.7 and 0.95mBar, respectively for the cycles B, C and D. The heating platetemperature was also increased from 5 to 50° C., but it was kept thesame for the three cycles B, C and D. Both pressure and heating platetemperature were kept constant during the drying process (A, B, C andD).

No significant difference was observed in acidification activity of FDproducts when dried with the mild (A) and aggressive freeze-dryingconditions (B-D). The time t_(a) was 91 min for the product obtainedwith the mild FD cycle compared to a t_(a) of 93-97 min for the FDproducts generated with the more aggressive cycles (B-D). Aggressive FDcycles (B-D) also resulted in a product with a pH-6h value in the samerange (4.9-5) to that of the mild FD cycle (pH-6h of 4.9).

Moreover, taking the precision of the analysis into consideration (ISO26323:2009), the acidification activity of the FD products was notsignificantly different when the FD product was obtained by the mildcycle or by the aggressive FD cycles. Thus, it can be concluded thataggressive drying at pressure in the range of 0.5-0.95 mBar exerts nodetrimental effect on the acidification activity of R-607-1.

Evaluation of freeze-dried products normally also includes theobservation of the product appearance after freeze-drying because one ofthe desired characteristic is to avoid changes in the freeze-driedproduct appearance. The product appearance after freeze-drying withaggressive drying cylcles (B-D) was assessed by visual comparison to areference freeze-dried product which was obtained by drying with themild FD cycle (A). No difference in products appearance was observed(Table 5). The fact that the FD product appearance and characteristicswere not changed by the aggressive drying procedures (B-D) was alsoconfirmed by a low value of water activity (a_(w)=0.03-0.05) of these FDproducts. This value was comparable to that of the FD product obtainedby the mild drying cycle (A) (0.03) (Table 5).

Despite the fact that no significant differences between acidificationactivity and a_(w) of FD products dried under aggressive or mild FDconditions were observed, a significant difference in the drying timewas seen (FIG. 16). More aggressive drying cycles (B-D) resulted inapproximately 3 times shorter drying times of between 11 and 14.5 hcompared to 36 h for the mild FD cycle (A). Thus, the more aggressivecycles result in a much more efficient drying process, especially withregard to the energy costs and the productivity.

The main conclusions from the results presented in Example 14, which wasperformed with the culture R-607-1, are:

Product quality and performance of the products dried under aggressiveconditions (0.5-0.95 mBar and 50° C.) were indistinguishable from thoseof product dried under ‘mild’ conditions (0.3 mBar and 5° C.).

-   -   No significant difference was observed in acidification activity        (t_(a) and pH-6h) of FD products when dried with the mild and        aggressive freeze-drying conditions.    -   All freeze-dried products from the aggressive dryings were well        dried and reached significantly lower water activity of less        than 0.05 as compared to 0.03 for that of the mild drying.    -   No difference in product appearance was seen when comparing FD        products obtained by the aggressive drying processes as compared        to that obtained by a mild drying process.    -   Aggressive drying processes resulted in an approximately 3 times        shorter drying time compared with that of the mild drying. This        results in a much more efficient drying process, especially        regarding the energy costs and productivity.

TABLE 5 Culture R-607-1: Comparison of product quality when freeze-driedunder mild (A) and aggressive conditions (B-D) with respect toacidification activity (t_(a), ph-6 h), water activity (a_(w)) andproduct appearance. A (5° C., 0.3 mBar); B (50° C., 0.5 mBar); C (50°C., 0.70 mBar); D (50° C., 0.95 mBar) FD Pressure Product appearance FDCycle (mBar) pH-6 h t_(a) a_(w) after freeze-drying A 0.3 4.9 91 0.03 0B 0.5 4.9 93 0.03 0 C 0.7 5.0 94 0.05 0 D 0.95 5.0 97 0.05 0

Example 15 Lactobacillus rhamnosus LGG®: Comparison of Product Qualitywhen Freeze-Dried Under Mild and Aggressive Conditions. Effect of HighTemperature (50-75° C.)

Lactobacillus rhamnosus (ATCC53103) was cultivated in 700 liters MRS.The bacteria in the fermentation broth were concentrated bycentrifugation using a centrifuge CSA-6 Westfalia. After centrifugation,the bacterial concentrate was mixed with cryoprotective solution (300 gto 1000 g cell concentrate). Afterwards, bacterial suspension was frozenwith liquid nitrogen in the form of pellets (i.e. PFD). Thecryoprotective solution consisted of skim milk (14%), monosodiumglutamate (6%) and water (80%). The frozen pellets of the bacterialsuspension are called pre-freeze-dried product (i.e. PFD). Pre-freezedried product (PFD) in the form of frozen pellets (i.e. PFD) with sizesof 1 to 5 mm in diameter were used. The PFD was kept at −50° C. untilbeing freeze-dried. The freeze-drying was carried out as described inExample 14 with four different freeze-drying (FD) cycles as describedbelow. Freeze-drying (FD) cycle A was a control cycle, i.e. a so-calledmild cycle with a constant heating plate temperature and constantchamber pressure during the drying process of 5° C. and 0.3 mBar,respectively. For the aggressive dryings (B-D), the pressure wasincreased from 0.3 mBar (A) to 0.5 mBar and kept constant during thedrying. The heating plate temperature was also increased from 5° C. to aconstant temperature of 50° C. and 75° C., respectively, for dryingcycles B and D. For cycle C the plate temperature was decreased from 75to 50° C. after 45% water removal.

Heating plate Freeze-drying Pressure temperature Comments cycle mBar °C. A (Control) 0.3  5° C. B 0.5 50° C. C 0.5 75° C. Heating platetemperature is lowered to +50° C. after 45% water is removed D 0.5 75°C.

The water activity (a_(w)) of freeze-dried products was measuredimmediately after freeze-drying. Water activity (a_(w)) measurement wasperformed as described in Example 1.

The number of viable cells after freeze-drying and after storage testwas determined as colony forming units (CFU) as described by Palmfeldtand Hahn-Hagerdal (2000), Int J Food Microbiol, 55(1-3):235-8. Theevaluation of the appearance of the freeze-dried product afterfreeze-drying and the categorization was performed according to thegrading described in Example 10.

Results

The aim of the present study was to investigate the effect of aggressivefreeze-drying conditions (high heating plate temperature) on thesurvival of freeze-dried Lactobacillus rhamnosus LGG®. Examination wasdone by comparison of viable cells (CFU) after freeze-drying of productsthat were freeze-dried with the mild cycle (A; 5° C. and 0.3 mBar) withproducts that were freeze-dried with the aggressive FD cycles (B-D;50-75° C. and 0.5 mbar). The results are summarized in Table 6.

No significant difference was seen in viable cell count afterfreeze-drying (CFU) when increasing the heating plate temperature from5° C. (A) to 50° C. (B) and 75° C. (C and D). Moreover, no visualstructural change occurred for FD product when the heating platetemperature was increased from 5° C. to 75° C.

In addition, the stability of the product, i.e. the cell survival, wasalso studied during storage for 3 weeks in open bags at 30° C. and 30%RH (Table 6). Surprisingly, the mild FD conditions (A; heating platetemperature of 5° C. and pressure of 0.3 mBar) affect the cell survivalnegatively during storage. This FD (A) cycle resulted in the highestcell loss, and therefore, in the lowest viable cell count of 10.8 (logCFU/g) compared to that of 11.5 (log CFU/g) for drying cycle B, and11-11.1 (log CFU/g) for drying cycles C and D, respectively. The resultswere also confirmed by flow cytometry (data not shown). Thus, it can beconcluded that aggressive drying cycles (B-D) with a heating platetemperature of 50 and 75° C. results in FD product with higher cellviability during storage compared to the mild FD conditions (A) with aheating plate temperature of 5° C.

Also, no visual change in the pellet structure was seen when the productwas freeze-dried under high temperatures of 50 and 75° C. (B-D) ascompared with a low temperature of 5° C. (A) (Table 6). The fact thatthere was no change in the pellet structure of the FD products thatunderwent aggressive drying cycles (B-D) was also confirmed by therather low water activity of the FD product of less than 0.03, which wasalso below the Limit of Detection (LOD) of 0.03 for the equipment.

In addition, the more aggressive drying cycles (B-D) resulted in 3 timesshorter drying times of 10-14.5 h compared with that of 36 h for themild FD cycle (A) (FIG. 17). Thus, these were much more efficient dryingprocesses, especially with regard to the energy cost and productivity.

The main conclusions from the results presented in Example 15 that wereperformed with the culture LGG® are the following:

-   -   Aggressive drying cycles (50 and/or 75° C. and 0.5 mBar)        resulted in FD products with a higher cell viability after        storage compared with the mild FD conditions (5° C. and 0.3        mBar), although the products showed a comparable viable cell        count after freeze-drying (CFU).    -   No difference in products appearance was seen when comparing FD        products obtained by aggressive drying cycles (50; 75° C. and        0.5 mBar) compared with that of the mild drying cycle (5° C. and        0.3 mBar). This is in agreement with the conclusions drawn in        Example 14 above.    -   Freeze-dried products from the aggressive drying cycles were        well dried. They reached the same water activity of less than        0.03 as those products of the mild drying. This was also below        the Limit of Detection (LOD) of 0.03 for the equipment. This is        in agreement with the conclusions drawn in Example 14.    -   Aggressive drying cycles resulted in approximately 3 times        shorter drying time as compared with that of the mild drying        cycle, which results in much more efficient drying processes,        especially regarding the energy costs and the productivity. This        is in agreement with the conclusions drawn in Example 14.

TABLE 6 Culture LGG ®: Comparison of the product quality of productsthat were freeze-dried under mild (A) or aggressive conditions (B-D)with regard to the water activity (a_(w)), product appearance and cellsurvival (CFU) after freeze- drying and 3 weeks storage at 30° C. and30% RH. A (5° C., 0.3 mBar); B (50° C., 0.5 mBar); C (75° C.→50° C., 0.5mBar); D (75° C., 0.5 mBar) Cell viability Product Temperature- Start:After 3 Cell loss after appearance Heating Water after FD weeks storageafter plates activity Log storage Log loss freeze- FD Profile (° C.) awCFU/g Log CFU/g CFU/g drying A (Control)  5 0.03 11.6 10.8 0.8 0 B 500.03 11.5 11.5 0.0 0 C 75 → 50 0.03 11.5 11.1 0.4 0 D 75 0.03 11.3 11.00.3 0

Example 16

-   -   Streptococcus thermophilus, ST-10255: Production Scale        Freeze-Drying at Aggressive Drying Conditions. Comparison of        Product Quality with a Reference Product from a Pilot Scale that        was Obtained by Mild Freeze-Drying Conditions.

Frozen pellets of Streptococcus thermophilus strain ST10255 in skim milkand monosodium glutamate with a size of 1 to 10 mm in diameter were usedfor the freeze-drying trials carried out at pilot scale as describedbelow. The frozen pellets were kept at −50° C. until being freeze-dried.Two dryings were carried out. One was done at production scale underaggressive drying condition, and the second one was done at pilot scaleunder mild freeze-drying conditions (the reference).

Production Scale Freeze-Drying

The freeze-drying was carried out at a production scale with the AtlasRay™ Batch Dryer concept with continuous De-Icing System (Atlas Ray™125-S; GEA Niro). The effective tray area is 114 m². Typical inputcapacity solids 15% (kg/24 hours) is 2965 which translates to outputcapacity (kg/24 hours) of 460 kg. The heating plates provide heatradiation to the trays. The maximum sublimation capacity (kgwater/hours) is 280. For more information see the brochure, Title:Freeze drying—Atlas RAY™ plants for the food and beverage industries,Brochure number: BNA 832, GEA Niro, GEA Process Engineering

The frozen pellets of ST-10255 were freeze-dried in 2 steps.

Primary drying of the material was performed at a pressure of 0.6 mBarand a heating plate temperature of 65° C. This step was performed for aperiod of time that lasted at least 10 h; or until at least 75% water isremoved; and the temperature of the material dos not exceed 35° C.

The secondary drying was performed at a temperature of 35° C. and thesame pressure of 0.6 mBar. The temperature of the material did notexceed 35° C. This step was performed for a period of 11 hours.

Pilot Scale Trial Freeze-Drying

As a reference, a drying cycle was performed at Pilot scale using thesame PFD product as used for the production scale trial. Freeze-dryingwas carried out as described in Example 14 with a freeze-drying cycleinvolving a constant heating plate temperature of 5° C. and a constantchamber pressure of 0.3 mBar.

The freeze-drying was performed in a Hetosicc freeze dryer, CD-10-1,Heto Lab equipment, Heto-Holten A/S, Allerod, Denmark, as described inExample 14.

The following freeze-drying cycles were used:

Load FD Profiles in FD Heating plate cabinet Pressure temperature Scale(kg) (mBar) (° C.) Comment Pilot 10 A mild 0.3 5 Production 2000 B 0.665 Secondary aggressive drying was done at 35° C.

Analysis

The water activity (a_(w)) of the freeze-dried products was measuredimmediately after freeze-drying. Water activity (a_(w)) measurement wasperformed as described in Example 1. The determination of theacidification activity (t_(a) and pH-6h) was performed as described inExample 14. The evaluation of the appearance of the freeze-dried productafter freeze-drying and the categorization was performed according thegrading described in Example 10.

Results

Two drying processes were carried out. One drying process was performedin a Pilot scale freeze-dryer using a mild freeze-drying cycle (A)operating at temperature of 5° C. and a pressure of 0.3 mBar. The seconddrying process was performed in a production scale freeze-dryer withaggressive conditions, i.e. with a heating plate temperature of 65° C.and a pressure of 0.6 mBar in the primary drying. Results from bothdryings are summarized in Table 7.

Taking the precisions of the analysis into consideration (ISO26323:2009), no significant difference was observed in acidificationactivity of the product that was dried under mild (A) conditions at thePilot scale compared with that dried under aggressive freeze-dryingconditions (B) at production scale.

-   -   t_(a): Time (t_(a)) was 47 min for the product obtained with the        mild FD cycle (A) compared to a t_(a) of 53 min for the        aggressive FD product (B) that was produced at production scale.    -   pH6h: The FD product from the aggressive cycle from the        production scale process had the same pH-6h as compared to the        product obtained by the mild FD cycle at pilot scale.

With respect to water activity and product appearance afterfreeze-drying:

-   -   No visual change in pellets structure was seen when the product        was freeze-dried at production scale with an aggressive        cycle (B) compared to the product dried at Pilot scale under        mild drying conditions (A) (Table 7).    -   A comparable water activity was obtained. The water activity of        the FD product that was produced with aggressive drying was 0.08        compared to that of 0.03 for the FD product that was obtained by        the mild drying cycle (A) at Pilot scale.

Overall, the conclusions from the results presented in Example 16carried out at production scale with culture ST-10255 are in agreementwith the conclusions drawn in Examples 14 (culture R-607-1) and 15(culture LGG®).

Product quality and performance of the products dried under aggressiveconditions (0.65 mBar and 60° C.) at production scale wereindistinguishable from product that was dried under mild conditions (0.3mBar and 5° C.) at pilot scale.

-   -   No significant difference was observed in the acidification        activity (t_(a) and pH-6h) of the FD product when dried with the        mild or aggressive freeze-drying conditions at Pilot and        Production scale, respectively.    -   The freeze-dried product from the aggressive drying process from        the production scale was well dried and reached a low water        activity of 0.08 as compared to 0.03 for that of the mild drying        at Pilot scale.    -   No difference in the product appearance was seen when comparing        the FD products obtained by the aggressive drying from        production scale compared to that of the mild drying from pilot        scale.

TABLE 7 Culture ST-10255: Comparison of the product quality whenfreeze-dried under mild (A) and aggressive conditions (B-D) with respectto the acidification activity (t_(a), ph-6 h), water activity (a_(w))and product appearance. A (5° C., 0.3 mBar); B (65° C. → 35° C., 0.6mBar). Freeze-dried product quality Product Scale FD profile pH-6 h TaWater activity appearance Pilot A (mild) 5.1 47 0.03 0 Production B 5.153 0.08 0 (aggressive)

Example 17 LH-B-02: Lactobacillus helveticus, Mixture of 4 SingleStrains of Lactobacillus helveticus (Mixture of CHCC2160, CHCC3204,CHCC3205 and CHCC3206)

Frozen pellets of Lactobacillus helveticus, mixture of 4 single strains(mixture of CHCC2160, CHCC3204, CHCC3205 and CHCC3206) in skim milk andmonosodium glutamate with a size of 1 to 5 mm in diameter were used forthe freeze-drying trials carried out at pilot scale as described inexample 14. The frozen pellets were kept at −50° C. until beingfreeze-dried. The frozen pellets of the bacterial suspension are calledpre-freeze-dried product (i.e. PFD).

The freeze-drying was carried out as described in Example 14 with fivedifferent freeze-drying (FD) cycles as described below. Freeze-drying(FD) cycle A was a control cycle, i.e. a so-called mild cycle with aconstant heating plate temperature and constant chamber pressure duringthe drying process of 5° C. and 0.3 mBar, respectively. For theaggressive dryings (B-E), the pressure was increased from 0.3 mBar (A)to 0.95 mBar (E) and kept constant during the drying. The heating platetemperature was also increased from 5° C. to a constant temperature of50° C. for drying cycles B, D and E. Both pressure and heating platetemperature were kept constant during the drying process (A, B, D andE). For cycle C the plate temperature was decreased from 75 to 50° C.after 45% water removal.

Heating plate Freeze-drying Pressure temperature cycle mBar ° C.Comments A (Control) 0.3  5° C. B 0.5 50° C. C 0.5 75° C. Heating platetemperature is lowered to +50° C. after 45% water is D 0.7 50° C. E 0.9550° C.

The water activity (a_(w)) of freeze-dried products was measuredimmediately after freeze-drying. Water activity (a_(w)) measurement wasperformed as described in Example 1.

The acidification activity in the freeze-dried culture was measuredaccording to Example 14. The evaluation of the appearance of thefreeze-dried product after freeze-drying and the categorization wasperformed according to the grading described in Example 10.

Results

The aim of the present study was to investigate the effect of aggressivefreeze-drying conditions (high pressure and high temperature) on thequality of freeze-dried products of LH-B 02 which is a mixed culture of4 single strains of Lactobacillus helveticus (mixture of CHCC2160,CHCC3204, CHCC3205 and CHCC3206). Examination was done by comparison ofboth water activity (a_(w)) and acidification activity (t_(a), pH-6h) offreeze-dried products obtained by aggressive (B-E) and mild (A) FDcycles (see table above). The mild drying (A, reference drying) wascarried out with a constant heating plate temperature of 5° C. and witha constant chamber pressure of 0.3 mBar. For the aggressive dryings(B-E), the pressure was increased from 0.3 mBar (A) to 0.5, 0.7 and 0.95mBar, respectively for the cycles B-C, D and E. The heating platetemperature was also increased from 5 to 50° C., but it was kept thesame for the three cycles B, D and E. Both pressure and heating platetemperature were kept constant during the drying process (A, B, D andE). For cycle C, the pressure was kept constant during the dryingprocess of 0.5 mBar, but the plate temperature was decreased from 75 to50° C. after 45% water removal.

No significant difference was observed in acidification activity of FDproducts when dried with the mild (A) and aggressive freeze-dryingconditions (B-E). The time t_(a) was 135 min for the product obtainedwith the mild FD cycle compared to t_(a) of 136-143 min for the FDproducts generated with the more aggressive cycles (B-E). Aggressive FDcycles (B-E) also resulted in a product with a pH-6h value in the samerange (5.6) to that of the mild FD cycle (pH-6h of 5.5).

Moreover, taking the precision of the analysis into consideration (ISO26323:2009), the acidification activity of the FD products was notsignificantly different when the FD product was obtained by the mildcycle or by the aggressive FD cycles. Thus, it can be concluded thataggressive drying at pressure in the range of 0.5-0.95 mBar exerts nodetrimental effect on the acidification activity of LH-B-02, which is amixed culture of 4 single strains of Lactobacillus helveticus.

TABLE 8 Culture LH-B 02: Comparison of product quality when freeze-driedunder mild (A) and aggressive conditions (B-E) with respect toacidification activity (t_(a), ph-6 h), water activity (a_(w)) andproduct appearance. A (5° C., 0.3 mBar); B (50° C., 0.5 mBar); C (75°C.→50° C., 0.5 mBar); D (50° C., 0.70 mBar); E (50° C., 0.95 mBar)Product appearance FD Profile pH 6 timer t_(a) a_(w) after freeze-dryingA (Control) 5.5 135 <0.03 0 B 5.6 142 <0.03 0 C 5.6 140 <0.03 0 D 5.6136 <0.03 0 E 5.6 141 <0.03 0

Evaluation of freeze-dried products normally also includes theobservation of the product appearance after freeze-drying because one ofthe desired characteristic is to avoid changes in the freeze-driedproduct appearance. The product appearance after freeze-drying withaggressive drying cylcles (B-E) was assessed by visual comparison to areference freeze-dried product which was obtained by drying with themild FD cycle (A). No difference in products appearance was observed(Table 8). The fact that the FD product appearance and characteristicswere not changed by the aggressive drying procedures (B-E) was alsoconfirmed by a low value of water activity (a, =0.03) of these FDproducts. This value was the same to that of the FD product obtained bythe mild drying cycle (A) (0.03) (Table 8).

The main conclusions from the results presented in Example 17, which wasperformed with the culture LH-B 02, which is a mixed culture of 4 singlestrains of Lactobacillus helveticus, are:

Product quality and performance of the products dried under aggressiveconditions (pressure 0.5-0.95 mBar and temperature 50° C. and 75° C.)were indistinguishable from those of product dried under ‘mild’conditions (0.3 mBar and 5° C.).

-   -   No significant difference was observed in acidification activity        (t_(a) and pH-6h) of FD products when dried with the mild and        aggressive freeze-drying conditions.    -   All freeze-dried products from the aggressive dryings were well        dried and reached significantly low water activity of less than        0.03, which was the same as that obtained by the mild drying        (0.03).    -   No difference in product appearance was seen when comparing FD        products obtained by the aggressive drying processes as compared        to that obtained by a mild drying process.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

REFERENCES

-   Ferreira, V. et al. “Survival of Lactobacillus sakei during heating,    drying and storage in the dried state when growth has occurred in    the presence of sucrose or monosodium glutamate.” Biotechnology    Letters 27(4) (2005): 249-52.-   de Valdez G F, et al. “Effect of Drying Medium on Residual Moisture    Content and Viability of Freeze-Dried Lactic Acid Bacteria.” Appl    Environ Microbiol. 49(2) (1985): 413-15.-   Kurtmann L, et al. “Water activity-temperature state diagrams of    freeze-dried Lactobacillus acidophilus (LA-5(D): Influence of    physical state on bacterial survival during storage.” Biotechnol.    Prog 25(1) (2009): 265-70.-   Laroche C, Fine F, and Gervais P. “Water activity affects heat    resistance of microorganisms in food powders.” International Journal    of Food Microbiology 97.3 (2005): 307-15.-   Patel S M, Doen T, Pikal M J. “Determination of End Point of Primary    Drying in Freeze-Drying Process Control.” AAPS PharmSciTech 11.1    (2010): 73-84.-   Stadhouders et al, “Preservation of starters and mass production of    starter bacteria”, Neth. Milk Dairy J. 23, 182-199. 1969.-   Fernanda et al. (2004), Collapse temperature of bacterial    suspensions: the effect of cell type and concentration. Cryoletters    25[6], 425-34.-   Palmfeldt J and Hahn-Hagerdal B. “Influence of culture pH on    survival of Lactobacillus reuteri subjected to freeze-drying.” Int J    Food Microbiol. 55.1-3 (2000): 235-38.-   PIKAL M. J.; SHAH S. The collapse temperature in freeze drying:    dependence on measurement methodology and rate of water removal from    the glassy phase. International Journal of Pharmaceutics. 62.    2-3 (1990) 165-186.-   Georges CORRIEU; Francois-Marie LUQUET, 2008; Bactéries lactiques:    De la génétique aux ferments-   Patent references: WO2006/125446, US7037708B1, WO99057242,    WO2012088261, WO2012076665, WO 2013/083762, WO2013/024178.

All references cited in this patent document are hereby incorporatedherein in their entirety by reference.

The invention claimed is:
 1. A process for drying a bacteria-containingconcentrate, comprising: (i) washing a bacteria-containing concentrateto obtain a washed bacteria-containing concentrate; (ii) freezing saidwashed bacteria-containing concentrate; and (iii) freeze-drying saidfrozen bacteria-containing concentrate obtained in step (ii) by atwo-step freeze-drying process comprising (a) a first freeze-drying stepcomprising freeze-drying under a pressure in the range of from 0.4 to0.65 mBar and a constant heating plate temperature in the range of from50 to 75° C., until from 40 to 80% of the water is removed; followed by(b) a second freeze-drying step comprising freeze-drying under apressure in the range of from 0.4 to 0.65 mBar and a constant heatingplate temperature in the range of from 35 to 50° C., until from 40 to99% of the water is removed.
 2. The process of claim 1, wherein thepressure in the first and second freeze-drying steps is in a rangeselected from the group consisting of 0.5 to 0.6 mBar, and 0.4 to 0.6mBar.
 3. The process of claim 1, wherein the pressure in the firstfreeze-drying step is kept within the range for a time period selectedfrom the group consisting of more than 1 hour, more than 2 hours, morethan 3 hours, more than 4 hours, more than 5 hours, and more than 7hours.
 4. The process of claim 1, wherein the constant heating platetemperature in the first freeze-drying step is in a range of from 60 to75° C.
 5. The process of claim 1, wherein the constant heating platetemperature in the first freeze-drying step is maintained for a timeperiod selected from the group consisting of more than 1 hour, more than2 hours, more than 3 hours, more than 4 hours, more than 5 hours, andmore than 7 hours.
 6. The process of claim 1, wherein at least 60%, atleast 75%, at least 80%, at least 90%, at least 95%, or at least 99% ofthe water is removed.
 7. The process of claim 1, wherein (a) the firstand second freeze-drying steps are performed in a freeze-dryerconfigured to process a batch size of more than 100 kg; and/or (b) thenumber of shelves and/or heating plates is more than 5; and/or (c) thethickness of the frozen bacteria-containing material on the producttrays at initiation of the freeze: drying process is from 8 to 60 mm;and/or (d) the bacteria-containing concentrate comprises an additiveselected from the group consisting of inosine, inositol, inosinemonophosphate (IMP), trehalose, sucrose, maltodextrin, yeast extract,skim milk powder, glutamate and protein hydrolyzate.
 8. The process ofclaim 1, wherein the bacteria-containing concentrate comprises one ormore additives selected from the group consisting of inosine, inositol,trehalose and sucrose, and wherein the first freeze-drying step isperformed under a pressure in the range of from 0.4 to 0.6 mBar for morethan 1 hour and/or until at least 75% of the water is removed.
 9. Theprocess of claim 1, wherein said bacteria-containing concentratecomprises bacteria from at least one lactic acid bacteria genus selectedfrom the group consisting of Lactococcus, Lactobacillus, Leuconostoc,Carnobacterium, Pediococcus, and Streptococcus.
 10. The process of claim1, wherein said bacteria-containing concentrate comprises bacteria fromat least one species selected from the group consisting of Leuconostocspp., Bifidobacterium ssp, Lactococcus lactis, Lactococcus cremoris,Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefir,Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus,Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillussalivarius, Lactobacillus curvatus, Lactobacillus bulgaricus,Lactobacillus sake, Lactobacillus reuteri, Lactobacillus lactis,Lactobacillus delbreuckii, Lactobacillus plantarum, and Streptococcusthermophiles.
 11. The process of claim 1, wherein the number of bacteriain the bacteria-containing concentrate is selected from the groupconsisting of from 10⁸ to 10¹³ cfu/mL, from 10⁹ to 10¹³ cfu/mL, from 10⁹to 10¹² cfu/mL, and from 10¹⁰ to 10¹² cfu/mL.
 12. The process of claim1, wherein the second freeze-drying step is performed until at least 90%of the water is removed.
 13. The process of claim 1, wherein the numberof bacteria in the bacteria-containing concentrate is from 10⁹ to 10¹²cfu/mL.
 14. The process of claim 1, wherein the washing comprises addingan aqueous solution to the bacteria-containing concentrate to obtain asuspension and concentrating the suspension to obtain a washedbacteria-containing concentrate.
 15. The process of claim 14, whereinthe volume of the added aqueous solution is from 0.3 liters to 10 litersper liter of the bacteria-containing concentrate.
 16. The process ofclaim 14, wherein the washed bacteria-containing concentrate has asolids content of from 5 to 20 percent.
 17. A process for drying abacteria-containing concentrate, comprising: (i) washing abacteria-containing concentrate to obtain a washed bacteria-containingconcentrate; (ii) freezing said washed bacteria-containing concentrate;and (iii) freeze-drying said frozen bacteria-containing concentrateobtained in step (ii) by a two-step freeze-drying process comprising (a)a first freeze-drying step comprising freeze-drying under a pressure inthe range of from 0.4 to 0.65 mBar and a constant heating platetemperature in the range of from 50 to 75° C., until the driedconcentrate contains at least 50% dry matter; followed by (b) a secondfreeze-drying step comprising freeze-drying under a pressure in therange of from 0.4 to 0.65 mBar and a constant heating plate temperaturein the range from of 35 to 50° C., until the dried concentrate containsat least 95% dry matter.
 18. The process of claim 17, wherein (a) thefirst and second freeze-drying steps are performed in a freeze-dryerconfigured to process a batch size of more than 100 kg; and/or (b) thenumber of shelves and/or heating plates is more than 5; and/or (c) thethickness of the frozen bacteria-containing material on the producttrays at initiation of the freeze-drying process is from 8 to 60 mm;and/or (d) the bacteria-containing concentrate comprises an additiveselected from the group consisting of inosine, inositol, inosinemonophosphate (IMP), trehalose, sucrose, maltodextrin, yeast extract,skim milk powder, glutamate and protein hydrolyzate.
 19. The process ofclaim 17, wherein said bacteria-containing concentrate comprisesbacteria from at least one lactic acid bacteria genus selected from thegroup consisting of Lactococcus, Lactobacillus, Leuconostoc,Carnobacterium, Pediococcus, and Streptococcus.